Mitofusin activators and methods of use thereof

ABSTRACT

Compositions including small molecule mitofusin activators are described. The mitofusin activators are useful for treating diseases or disorders associated with a mitochondria-associated disease, disorder, or condition such as diseases or disorders associated with mitofusin 1 (MFN1) and/or mitofusin 2 (MFN2), or mitochondrial dysfunction. Methods of treatment, pharmaceutical formulations, and screening methods for identifying compounds that activate mitochondrial fusion are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/087,971, filed on Nov. 3, 2020, which is a continuation ofU.S. Patent Application 16/935; 489, filed on Jul. 22, 2020; which acontinuation-in-pail of International Patent ApplicationPCT/US2019/046356, filed on Aug. 13, 2019, which claims the benefit ofpriority under 35 U.S.C. § 119 from U.S. Provisional Patent Application62/797,513, filed on Jan. 28, 2019, each of which is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to compositions and methods fortreating genetic and traumatic neurodegenerative diseases, disorders, orconditions. Also provided are methods for high-throughput screening ofcompositions.

SUMMARY

Among the various aspects of the present disclosure is the provision ofnovel chemical classes of small molecule mitofusin activators andmethods of use thereof.

One aspect of the present disclosure provides for methods of treatingneurodegenerative diseases, disorders, or conditions. In some features,the method comprises administering to a subject a therapeuticallyeffective amount of a composition of one or more mitofusin activators orpharmaceutically acceptable salts thereof; the mitofusin activatorsstimulate mitochondrial fusion and subcellular mitochondrial transport.

Another aspect of the present disclosure provides for a method ofactivating mitofusin in a subject in need thereof. In some features, themethod comprises administering to a subject a composition of one or moremitofusin activators or pharmaceutically acceptable salts thereof; themitofusin activator stimulates mitochondrial fusion and subcellulartransport; the subject has a genetic or traumatic neurodegenerativedisease, disorder, or condition; the mitofusin activator is not acompound selected from the following compounds:

Another aspect of the present disclosure provides for methods ofenhancing damaged nerve repair or regeneration in a subject in needthereof. In some features, the method comprises administering to asubject a composition comprising one or more mitofusin activators orpharmaceutically acceptable salts thereof; the mitofusin activatorregulates mitochondrial fusion and subcellular transport; the subjecthas genetic neurodegeneration or traumatic nerve injury; the mitofusinactivator is not a compound selected from the following compounds:

In some aspects, the mitofusin activator: has substantially betterfunctional potency of both1-[2-(benzylsulfanyl)ethyl]-3-(2-methylcyclohexyl)urea (Cpd A, RochaScience 2018) and2-{2-[(5-cyclopropyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]propanamido}-4H,5H,6H-cyclopenta[b]thiophene-3-carboxamide(Cpd B, Rocha Science 2018); or stimulates mitofusin activity (e.g.,mitochondrial fusion and subcellular transport).

In some aspects, the mitofusin activator: enhances mitochondrialtransport in nerve axons; increases mitochondrial polarization; correctscell and organ dysfunction caused by primary abnormalities inmitochondrial fission or fusion; corrects cell and organ defects inwhich secondary mitochondrial dysfunction is a contributing factor;reverses mitochondrial defects (e.g., dysmorphometry, clustering, lossof polarization, loss of motility); restores, activates, regulates,modulates, promotes, or enhances the fusion, function, tethering,transport, trafficking (e.g., axonal mitochondrial trafficking),mobility, or movement of mitochondria (in, optionally, a nerve or aneuron); enhances mitochondrial elongation or mitochondrial aspectratio; disrupts intramolecular restraints in MFN2; allostericallyactivates MFN2; corrects mitochondrial dysfunction and cellulardysfunction; repairs defects in neurons with mitochondrial mutations; ortargets MFN1 or MFN2.

In some aspects, the mitofusin activator comprises one or more compoundshaving structures represented by formulas (I) or (II):

-   -   or a pharmaceutically acceptable salt, tautomer, or stereoisomer        thereof wherein R¹ is a non-, mono-, or poly-substituted aryl,        C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, or C₃₋₈ heterocyclyl, and R²        is a non-, mono-, or poly-substituted aryl, C₃₋₈ cycloalkyl,        C₃₋₈ heteroaryl, or C₃₋₈ heterocyclyl.

In some aspects, the mitofusin activator is selected from a compoundhaving a structure represented by formulas (I) or (II)

wherein R¹ is selected from the following moieties:

-   -   and R² is selected from the following moieties:

In some aspects, R¹ and R² are optionally substituted by one or more of:acetamide, C₁₋₈ alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl,Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl,hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone,and/or thiophene; and optionally further substituted with one or moreacetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl,cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F,halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/orthiophene. Optionally the aforementioned alkyl, cycloalkyl, heteroaryl,heterocyclyl, indole, or phenyl is further substituted with one or moreof the following: acetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl,carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S,sulfoxide, sulfone, and/or thiophene.

In some aspects, the compound is selected from the following moieties:

Yet another aspect of present disclosure provides for a pharmaceuticalcomposition comprising a mitofusin activator, optionally in combinationwith one or more therapeutically acceptable diluents or carriers.

In some aspects, the pharmaceutical composition comprises apharmaceutically acceptable excipient.

In some aspects, the pharmaceutical composition comprises at least onecompound selected from neuroprotectants, anti-Parkinsonian drugs,amyloid protein deposition inhibitors, beta amyloid synthesisinhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs,anti-amyotrophic lateral sclerosis drugs, anti-Huntington's drugs,anti-Alzheimer's drugs, anti-epileptic drugs, and/or steroids.

Yet another aspect of the present disclosure provides for a method oftreating a mitochondria-associated disease, disorder, or condition in asubject, the method comprising administering to the subject atherapeutically effective amount of a mitofusin activator.

In some aspects, the subject is diagnosed with or is suspected of havinga mitochondria-associated disease, disorder, or condition. In someaspects, the mitochondria-associated disease, disorder, or condition isone or more of: a central nervous system (CNS) or peripheral nervoussystem (PNS) injury or trauma, such as trauma to the CNS or PNS, crushinjury, spinal cord injury (SCI), traumatic brain injury, stroke, opticnerve injury, or related conditions that involve axonal disconnection; achronic neurodegenerative condition wherein mitochondrial fusion,fitness, or trafficking are impaired; a disease or disorder associatedwith mitofusin 1 (MFN1) or mitofusin 2 (MFN2) or mitochondrialdysfunction, fragmentation, or fusion; dysfunction in MFN1 or MFN2unfolding; mitochondria dysfunction caused by mutations; a degenerativeneurological condition, such as Alzheimer's disease, Parkinson'sdisease, Charcot-Marie-Tooth disease, or Huntington's disease;hereditary motor and sensory neuropathy, autism, autosomal dominantoptic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber'shereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosingencephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis(NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, and/or pyruvate dehydrogenase complex deficiency(PDCD/PDH).

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 illustrates a structural model of human MFN2 in theclosed/inactive (left) and open/active conformation (right) that ispromoted by mitofusin activators.

FIG. 2 depicts a series of chemical structures for a new class ofurea-based mitofusin activators and a dot plot graph that shows thefunction of each new compound expressed as the mitofusin-dependentmitochondrial elongation provoked by each in comparison with theprototype of the previously described class of small molecule mitofusinactivators, Reg C.

FIG. 3 depicts a series of chemical structures for yet another new classof amide-based mitofusin activators and a dot plot graph that shows thefunction of each new compound expressed as the mitofusin-dependentmitochondrial elongation provoked by each in comparison with theprototype of the previously described class of small molecule mitofusinactivators, Reg C.

FIG. 4 depicts a series of chemical structures for yet another new classof amide-based cyclic backbone mitofusin activators and a dot plot graphthat shows the function of each new compound expressed as themitofusin-dependent mitochondrial elongation provoked by each incomparison with the prototype of the previously described class of smallmolecule mitofusin activators, Reg C.

FIG. 5 depicts a series of chemical structures for yet another new classof amide-based mitofusin activators containing a heteroaromatic groupand a dot plot graph that shows the function of each new compoundexpressed as the mitofusin-dependent mitochondrial elongation provokedby each in comparison with the prototype of the previously describedclass of small molecule mitofusin activators, Reg C.

FIG. 6A shows the dose-response relations of MiM5, MiM11, and MiM111compared to a prototype Chimera compound described in Rocha et alScience 2018; FIG. 6B shows MiM111 conformational opening of MFN 2mimics that of an agonist peptide described in Franco et al Nature 2016;and FIG. 6C shows that MiM111 promotes regrowth of mouseCharcot-Marie-Tooth 2A dorsal root ganglion neurons in culture and mayreverse disease in mouse model of Charcot-Marie-Tooth disease type 2A.

FIG. 7A illustrates an example of MiM111 reversing defects inexperimental CMT2A in a graph where the schematics at top showexperimental design and the dot blots at bottom show results; FIG. 7Bshows the results, in which MiM111 reversed a Rotarod defect in alltreated mice within 8 weeks of treatment (statistics used 2-way ANOVA).

FIG. 8 is a schematic depiction of a method for measuringmitofusin-dependent mitochondrial fusion in a manner suitable forhigh-throughput screening or detailed quantitative analysis of mitofusinactivator activity.

FIG. 9 shows results of mitochondrial fusion screening assay using themethods illustrated in FIG. 8.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery thatpharmacophore modeling of function-critical MFN2-derived interactingpeptides may produce small molecule peptidomimetic activators useful totreat mitochondrial-associated diseases, disorders, and conditions. Asshown herein, the present disclosure provides new chemical classes ofcompositions for regulating mitochondrial function. These compositionsmay be useful to correct cell and organ dysfunction caused by primaryabnormalities in mitochondrial fission, fusion and subcellularmotility/distribution, or in which secondary mitochondrial abnormalitiescontribute to disease.

Mitofusin Activators

The present disclosure provides for a new structurally-distinct class ofsmall molecule inhibitors of a function-critical MFN2 peptide-peptideinteraction. As described herein, a composition for the treatment of amitochondria-associated disease, disorder, or condition may comprise amitofusin activator, such as a peptidomimetic (e.g., a small-moleculethat mimics the chemico-structural features of a peptide). Apeptidomimetic may be a chemical peptidomimetic. For example, thepeptidomimetic may mimic a mitofusin-derived mini-peptide.

As described herein, a new generation of peptidomimetic small moleculeshas been developed. These compounds activate mitochondrial fusion bydirecting MFN1 and MFN2 to different conformational states. The firstsmall molecule peptidomimetics to target MFN1 or MFN2 (described inRocha et al. Science, 2018) had poor pharmacokinetic characteristics,making them “undruggable.” Described herein are members of astructurally distinct class of small molecule mitofusin activators thatactivate mitochondrial fusion and subcellular transport, have favorablepharmacokinetic properties, and may be used to correct mitochondrial andcellular dysfunction.

Mitofusin activators enhance mitochondrial elongation. Mitochondrialelongation may be measured by mitochondrial aspect ratio, but doing sois a time-consuming and indirect means of measuring mitofusin activatoractivity. Mitofusin activator activity is best measured throughdetermining the rate of mitochondria to fuse, or to exchange contents.As described herein, a method of high-throughput assaying of smallmolecules, peptides, or other bioactive compounds for mitochondrialfusion may be used to screen compounds for mitofusin activator activity.

Mitofusin Mini-Peptide

As described herein, a peptide mitofusin activator may be anMFN2-derived mini-peptide as described in Franco et al. Nature 2016.

Mfn Activator (Fusion-Promoting) Peptidomimetic

As described herein, a peptidomimetic may be a MFN activator(fusion-promoting) peptidomimetic that competes with endogenous MFN1 orMFN2 HR1-HR2 peptide-peptide interactions as described in Franco et al.Nature 2016 and Rocha et al. Science 2018.

Mitofusin activators may include the following compounds:

Mitofusin Activators: Structurally Distinct Small Molecules thatActivate Mfn1 and/or Mfn2

The small molecule mitofusin activators described herein are allostericmitofusin activators derived from the pharmacophore HR1-HR2peptide-peptide interaction model described in Rocha et al. Science2018, but which are structurally distinct and of separate chemicalclasses from those previously described. An activator is a substancethat partially or fully activates the protein to which it binds.

The mitofusin activators of the present disclosure may be of the formula(I) or (II):

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof, wherein R¹ is selected from the following moieties:

and wherein R² is selected from the following moieties:

Optionally, R¹ or R² in formula (I) or (II) may be independentlysubstituted by one or more of the following groups: acetamide, C₁₋₈alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/or thiopheneand optionally further substituted with acetamide, alkoxy, amino, azo,Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O,phenyl, S, sulfoxide, sulfone, and/or thiophene and the alkyl,cycloalkyl, heteroaryl, heterocyclyl, indole, or phenyl is optionallyfurther substituted with one or more of the following groups: acetamide,alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, and/or thiophene.

Optionally, the R¹ and R² groups in formula (I) or (II) may beindependently substituted with one or more of the following groups:hydroxyl; C₁₋₁₀ alkyl hydroxyl; amine; C₁₋₁₀ carboxylic acid;C₁₋₁₀carboxyl; straight chain or branched C₁₋₁₀ alkyl, optionallycontaining unsaturation; a C₂₋₈ cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; straight chain or branchedC₁₋₁₀ alkyl amine; heterocyclyl; heterocyclic amine; and/or arylcomprising phenyl, heteroaryl containing from one to four of thefollowing heteroatoms: N, O, and/or S, unsubstituted phenyl ring,substituted phenyl ring, unsubstituted heterocyclyl, and substitutedheterocyclyl. Optionally, the unsubstituted phenyl ring or substitutedphenyl ring may be independently substituted with one or more of thefollowing groups: hydroxyl; C₁₋₁₀ alkyl hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀ carboxyl; straight chain or branched C₁₋₁₀ alkyl,optionally containing unsaturation; straight chain or branched C₁₋₁₀alkyl amine, optionally containing unsaturation; a C₂₋₁₀ cycloalkyloptionally containing unsaturation or one oxygen or nitrogen atom;straight chain or branched C₁₋₁₀ alkyl amine; heterocyclyl; heterocyclicamine; and/or aryl comprising phenyl and heteroaryl containing from oneto four of the following heteroatoms: N, O, and/or S. Optionally, theunsubstituted heterocyclyl or substituted heterocyclyl may beindependently substituted with one or more of the following groups:hydroxyl; C₁₋₁₀ alkyl hydroxyl; amine; C₁₋₁₀ carboxylic acid; C₁₋₁₀carboxyl; straight chain or branched C₁₋₁₀ alkyl optionally containingunsaturation; straight chain or branched C₁₋₁₀ alkyl amine optionallycontaining unsaturation; a C₂₋₈ cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; heterocyclyl; straightchain or branched C₁₋₁₀ alkyl amine; heterocyclic amine; and/or arylcomprising a phenyl and a heteroaryl containing from one to four of thefollowing heteroatoms: N, O, and S. Any of the above may be furtheroptionally substituted.

In some aspects, R¹ or R² in formula (I) or (II) are optionallysubstituted by one or more of the following groups: acetamide, alkoxy,amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfone, and/or thiophene;and optionally further substituted with one or more of the followinggroups: acetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl,carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S,sulfoxide, sulfone, or thiophene; wherein the alkyl, cycloalkyl,heteroaryl, heterocyclyl, indole, or phenyl, is optionally furthersubstituted with one or more of acetamide, alkoxy, amino, azo, Br, C₁₋₈alkyl, carbonyl, carboxyl, Cl, cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl,C₃₋₈ heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S,sulfoxide, sulfone, and/or thiophene.

In another aspect of the disclosure, the mitofusin activator may be ofthe formula (III):

or a pharmaceutically salt thereof.

In Formula (III), o may be 0, 1, 2, 3, 4, or 5; p may be 0 or 1; and qmay be 0, 1, 2, 3, 4, or 5 with the proviso that the sum of o+p+q is notless than 3 or greater than 7; X may be cycloalkyl, heterocycloalkyl,aryl, or heteroaryl; Z may be cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; R^(a) and R^(b) may be independently H, F, alkyl, or C₃₋₇cycloalkyl, or R^(a) and R^(b) taken together may form a C₃₋₇ cycloalkylor heterocycloalkyl; R^(c) and R^(d) may be independently H, F, alkyl,COR^(g), or C₃₋₇ cycloalkyl, or R^(c) and R^(d) taken together may forma C₃₋₇ cycloalkyl or heterocycloalkyl; Y may be O, CR^(e)R^(f),CR^(g)═CR^(h), C≡C cycloalkyl, heterocycloalkyl, aryl, heteroaryl,NR^(g), S, SO₂, SONR^(h), NR^(h)SO₂, NR^(g)CO, CONR^(g), orNR^(g)CONR^(h); R^(e) and R^(f) may independently be H, F, alkyl, orcycloalkyl, or R^(e) and R^(f) may be taken together to form C₃₋₇cycloalkyl or heterocycloalkyl; R^(g) may be H, alkyl, or C₃₋₇cycloalkyl; and R^(h) may be H, alkyl, or C₃₋₇ cycloalkyl.

In some aspects, in the mitofusin activator of formula (III), o is 0, 1,2, 3, 4, or 5; p is 0 or 1; and q is 0, 1, 2, 3, 4, or 5 with theproviso that the sum of o+p+q is not less than 3 or greater than 7; X iscycloalkyl or heterocycloalkyl; Z is cycloalkyl, heterocycloalkyl, aryl,or heteroaryl; Y is O, CR^(e)R^(f), cycloalkyl, or aryl; and R^(a),R^(h), R^(c), R^(d), R^(e), R^(f), and R^(g) are independently H oralkyl.

In some aspects, in the mitofusin activator of formula (III), o may be0, 1, 2, 3, 4, or 5; p may be 0 or 1; and q may be 0, 1, 2, 3, 4, or 5with the proviso that the sum of o+p+q is not less than 3 or greaterthan 5; X may be a cycloalkyl having one to three of the followingsubstituents: R^(g), OR^(g), NR^(g)R^(h), F, or CF₃, or X may be aheterocycloalkyl containing one to two of the following optionallysubstituted heteroatoms: NR^(g), O, and S; Z may be aryl or heteroaryl;Y may be O, CH₂, or cycloalkyl; R^(a), R^(h), R^(c) and R^(d) may eachbe H; R^(g) may be H, alkyl, or C₃₋₇ cycloalkyl; and R^(h) may be H,alkyl, or C₃₋₇ cycloalkyl.

In some aspects, in the mitofusin activator of formula (III), o may be0, 1, 2, or 3; p may be 1; and q may be 0, 1, 2, or 3 with the provisothat the sum of o+p+q is not less than 3 or greater than 5; X may be acycloalkyl with one to three of the following substituents: R^(g),OR^(g), NR^(g)R^(h), F, or CF₃ or X may be a heterocycloalkyl having oneto two of the following optionally substituted heteroatoms: O, NR^(g),and S; Z may be aryl or heteroaryl; Y may be cyclopropyl or cyclobutyl;R^(a), R^(b), R^(c) and R^(d) may each be H; R^(g) and R^(h) mayindependently be H, alkyl, or C₃₋₇ cycloalkyl, or R^(g) and R^(h) takentogether may form a C₃₋₇ cycloalkyl.

In some aspects, in the mitofusin activator of formula (III), o may be0, 1, 2, 3, or 4; p may be 1; and q may be 0, 1, 2, 3, or 4 with theprovision that the sum of o+p+q is 5; X may be cycloalkyl having one tothree of the following substituents: R^(g), OR^(g), NR^(g)R^(h), F, andCF₃, or X may be a heterocycloalkyl containing one to two of thefollowing optionally substituted heteroatoms: O, NR^(g), and S; Z may bearyl or heteroaryl; Y may be O or CH₂; R^(a), R^(b), R^(c), and R^(d)may each be H; R^(g) may be selected from H, alkyl, and C₃₋₇ cycloalkyl;R^(h) may be selected from H, alkyl, COR^(g) and C₃₋₇ cycloalkyl, oroptionally R^(g) and R^(h) taken together may form a C₃₋₇ cycloalkyl.

In some aspects, in the mitofusin activator of formula (III), X may be4-hydroxycyclohexyl, 4-aminocyclohexyl, 4-(N-methyl)aminocyclohexyl,4-(N,N-dimethyl)aminocyclohexyl, 4-(N-acetylamino)cyclohexyl,4,4-difluorocyclohexyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperidinyl, N-methyl-piperidinyl, or N-acetyl-piperidinyl; Z may bearyl or heteroaryl; Y may be O or CH₂; R^(a), R^(b), R^(c), and R^(d)may each be H; o may be 0, 1, 2, 3, or 4; p may be 1; and q may be 0, 1,2, 3, or 4 with the proviso that the sum of o+p+q is 5.

In some aspects, in the mitofusin activator of formula (III), X iscycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each containing oneto four of the following heteroatoms: N, O, and S and having from zeroto four of the following substituents: R^(g), OR^(g), Cl, F, CN, CF₃,—NR^(g)R^(h), SO₂NR^(g)R^(h), NR^(g)SO₂R^(i), SO₂R^(i), CONR^(g)R^(h),NR^(h)COR^(i), C37 cycloalkyl, and heterocycloalkyl; Z is phenyl orheteroaryl, wherein the heteroaryl may contain from one to four atomsindependently selected from N, O, and S, and wherein the phenyl orheteroaryl has zero to four of the following substituents independentlyselected from R^(g), OR^(g), Cl, F, CN, CF₃, NR^(g)R^(h),SO₂NR^(g)R^(h), NR^(g)SO₂R^(i), SO₂R^(i), CONR^(g)R^(h), NR^(h)COR^(i),C₃₋₇ cycloalkyl, and/or heterocycloalkyl; Y is O or CH₂; R^(a), R^(b),R^(c), and R^(d) may each be H; R^(g) is selected from H, alkyl, andC₃₋₇ cycloalkyl; R^(h) is selected from H, alkyl, COR^(g) and C₃₋₇cycloalkyl, or R^(h) and R^(h) taken together may form a C₃₋₇cycloalkyl; R^(i) is alkyl or C₃₋₇ cycloalkyl; o is 0, 1, 2, 3, or 4; pis 1; and q is 0, 1, 2, 3, or 4 with the proviso that the sum of o+p+qis 5.

In some aspects, in the mitofusin activator of formula (III), X may be4-hydroxycyclohexyl, 4-aminocyclohexyl, 4-(N-methyl)aminocyclohexyl,4-(N,N-dimethyl)aminocyclohexyl, 4-(N-acetylamino)cyclohexyl,4,4-difluorocyclohexyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperidinyl, N-methyl-piperidinyl, or N-acetyl-piperidinyl; Z may bephenyl or heteroaryl, wherein the heteroaryl may contain one to three ofthe following heteroatoms: N, O, and S and wherein the phenyl orheteroaryl may contain zero to three of the following substituentsindependently selected from R^(g), OR^(g), Cl, F, CN, CF₃, NR^(g)R^(h),SO₂R^(i), CONR^(g)R^(h), NR^(h)COR^(i), C₃₋₇ cycloalkyl, and/orheterocycloalkyl; Y may be O or CH₂; R^(a), R^(b), R^(c), and R^(d) mayeach be H; R^(g) may be selected from H, alkyl, and C₃₋₇ cycloalkyl;R^(h) may be selected from H, alkyl, COR^(g) and C₃₋₇ cycloalkyl, orR^(g) and R^(h) taken together may form a C₃₋₇ cycloalkyl; R^(i) may beselected from alkyl and C₃₋₇ cycloalkyl; o may be 0, 1, 2, 3, or 4; pmay be 1; and q may be 0, 1, 2, 3, or 4 with the proviso that the sum ofo+p+q is 5.

In some aspects, in the mitofusin activator of formula (III), X may be4-hydroxycyclohexyl, 4-aminocyclohexyl, 4-(N-methyl)aminocyclohexyl,4-(N,N-dimethyl)aminocyclohexyl, 4-(N-acetylamino)cyclohexyl,4,4-difluorocyclohexyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperidinyl, 4-N-methyl-piperidinyl, or 4-N-acetyl-piperidinyl; Z may bephenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 6-pyrimidinyl,5-pyrimidinyl, 4-pyrimidinyl, or 2-pyrimidinyl, wherein the phenyl,pyridinyl, and pyrimidinyl moiety may have zero to two of the followingsubstituents independently selected from R^(g), OR^(g), Cl, F, CN, CF₃,NR^(g)R^(h), SO₂R^(i), CONR^(g)R^(h), and/or NR^(g)COR^(i); Y may be Oor CH₂; R^(a), R^(b), R^(c), and R^(d) may each be H; R^(g) may beselected from H, alkyl, and C₃₋₇ cycloalkyl; R^(h) may be selected fromH, alkyl, COR^(g) and C₃₋₇ cycloalkyl, or optionally, R^(g) and R^(h)taken together may form a C₃₋₇ cycloalkyl; R^(i) may be alkyl or C₃₋₇cycloalkyl; o may be 0, 1, 2, 3, or 4; p may be 1; and q may be 0, 1, 2,3, or 4 with the proviso that the sum of o+p+q is 5.

In some aspects, in the mitofusin activator of formula (III), X iscycloalkyl or heterocycloalkyl; Y is O, CR^(e)R^(f), cycloalkyl, oraryl; y is 1 R^(a), R^(b), R^(c), R^(e), and R^(f) are each H or alkyl;and p is 1. In more particular aspects, X is selected from a cycloalkylhaving one, two or three substituents independently selected from R^(g),OR^(g), NR^(g)R^(h), F, and CF₃, and a heterocycloalkyl containing oneor two optionally substituted heteroatoms independently selected from O,NR^(g), and S; Y is O, CH₂ or cycloalkyl, particularly cyclopropyl orcyclobutyl; R^(a), R^(b), R^(c), and R^(d) are each H, R^(g) and R^(h)are selected as above, or R^(g) and R^(h) taken together form aC₃₋₇-cycloalkyl, and p is 1.

In some aspects, in the mitofusin activator of formula (III), X may be acycloalkyl with having one, two, or three substituents independentlyselected from R^(g), OR^(g), NR^(g)R^(g), F, and CF₃, or aheterocycloalkyl containing one or two optionally substitutedheteroatoms independently selected from O, NR^(g), and S; Z is phenyl,2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl; 4-pyrimidinyl,5-pyrimidinyl, or 6-pyrimidinyl; Y is NR^(g); R^(c) and R^(d) are H; ois 0; p is 1; and q is 4. In more particular aspects, R^(g) is H. Inanother particular aspect, R^(g) is H and Z is phenyl or substitutedphenyl.

In some aspects, in the mitofusin activator of formula X may be acycloalkyl with having one, two, or three substituents independentlyselected from R^(g), OR^(g), NR^(g)R^(g), F, and CF₃, or aheterocycloalkyl containing one or two optionally substitutedheteroatoms independently selected from O, NR^(g), and S; Z is phenyl,2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl; 4-pyrimidinyl,5-pyrimidinyl, or 6-pyrimidinyl; R^(c) and R^(d) are H; o is 0; p is 0;and q is 5. In more particular aspects, Z is phenyl or substitutedphenyl.

In some aspects, in the mitofusin activator of formula X may be acycloalkyl with having one, two, or three substituents independentlyselected from R^(g), OR^(g), NR^(g)R^(g), F, and CF₃, or aheterocycloalkyl containing one or two optionally substitutedheteroatoms independently selected from O, NR^(g), and S; Z is phenyl,2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl; 4-pyrimidinyl,5-pyrimidinyl, or 6-pyrimidinyl; Y is cyclopropyl or cyclobutyl; R^(c)and R^(d) are H; o is 0 or 1; p is 1; and q is 1, 2 or 3. In moreparticular aspects, Z is phenyl or substituted phenyl. In some or otherparticular aspects, Y is cyclopropyl, particularly 1,2-cyclopropyl, andq is 2 or 3; or Y is cyclobutyl, particularly 1,3-cyclobutyl, and q is 1or 2.

In some aspects, in the mitofusin activator of formula X may be acycloalkyl with having one, two, or three substituents independentlyselected from R^(g), OR^(g), NR^(g)R^(g), F, and CF₃, or aheterocycloalkyl containing one or two optionally substitutedheteroatoms independently selected from O, NR^(g), and S; Z is phenyl,2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrimidinyl; 4-pyrimidinyl,5-pyrimidinyl, or 6-pyrimidinyl; Y is cyclopropyl or cyclobutyl; R^(a),R^(b), R^(c) and R^(d) are H; o is 0 or 1; p is 1; and q is 1, 2 or 3.In more particular aspects, Z is phenyl or substituted phenyl. In someor other particular aspects, Y is cyclopropyl, particularly1,2-cyclopropyl, and q is 2 or 3; or Y is cyclobutyl, particularly1,3-cyclobutyl, and q is 1 or 2. In some or other more particularaspects, Y is cyclobutyl, particularly 1,3-cyclobutyl, o is 1 and q is1.

In another aspect of the present disclosure, a method of treating adisease for which a mitofusin activator is indicated may compriseadministering to a mammal in need thereof a therapeutically effectiveamount of a compound of Formula (III)

or a pharmaceutically salt thereof. In Formula (III), o is 0, 1, 2, 3,4, or 5; p is 0 or 1; and q is 0, 1, 2, 3, 4, or 5 with the proviso thatthe sum of o+p+q is not less than 3 or greater than 7; X may becycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Z may be cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; R^(a) and R^(a) may independentlybe H, F, alkyl, or C₃₋₇ cycloalkyl, or R¹ and R² taken together may forma C₃₋₇ cycloalkyl or heterocycloalkyl; R^(c) and R^(d) may independentlybe H, F, alkyl, COR^(g), and/or C₃₋₇ cycloalkyl, or R^(c) and R^(d) maybe taken together to form a C₃₋₇ cycloalkyl or heterocycloalkyl; Y is O,CR^(e)R^(f), CR^(g)═CR^(h), CC cycloalkyl, heterocycloalkyl, aryl,heteroaryl, NR^(g), S, SO₂, SONR^(h), NR^(h)SO₂, NR^(g)CO, CONR^(g), orNR^(g)CONR^(h); R^(e) and R^(f) may independently be H, F, alkyl, and/orcycloalkyl, or R^(e) and R^(f) taken together may form a C₃₋₇ cycloalkylor heterocycloalkyl; and R^(g) and R^(h) may independently be H, alkyl,and/or C₃₋₇ cycloalkyl.

In some aspects, in the method of treating a disease for which amitofusin activator is indicated, the PNS or CNS disorder may beselected from any one or a combination of: a chronic neurodegenerativecondition wherein mitochondrial fusion, fitness, or trafficking areimpaired; a disease or disorder associated with mitofusin 1 (MFN1) ormitofusin 2 (MFN2) dysfunction; a disease associated with mitochondrialfragmentation, dysfunction, or dysmotility; a degenerative neuromuscularcondition such as Charcot-Marie-Tooth disease, amyotrophic lateralsclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson'sdisease, hereditary motor and sensory neuropathy, autism, autosomaldominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig's disease,cancer, mitochondrial myopathy, diabetes mellitus and deafness (DAD),Leber's hereditary optic neuropathy (LHON), Leigh syndrome, subacutesclerosing encephalopathy, neuropathy, ataxia, retinitis pigmentosa, andptosis (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE),myoclonic epilepsy with ragged red fibers (MERRF), mitochondrialmyopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms(MELAS), mtDNA depletion, mitochondrial neurogastrointestinalencephalomyopathy (MNGIE), dysautonomic mitochondrial myopathy,mitochondrial channelopathy, or pyruvate dehydrogenase complexdeficiency (PDCD/PDH), diabetic neuropathy, chemotherapy-inducedperipheral neuropathy, crush injury, SCI, traumatic brain injury (TBI),stroke, optic nerve injury, and/or related conditions that involveaxonal disconnection.

In some aspects of the disclosure, in the method of treating a diseasefor which a mitofusin activator is indicated, the composition mayfurther comprise a pharmaceutically acceptable excipient.

In some aspects of the disclosure, in the method of treating a CNSand/or PNS genetic and/or non-genetic neurodegenerative condition,injury, damage, and/or trauma comprising administering to the subject atherapeutically effective amount of a mitofusin activator according tothe present disclosure.

In some aspects of the disclosure, in the method of treating a CNS orPNS genetic or non-genetic neurodegenerative condition, injury, damage,or trauma, the subject may be diagnosed with or is suspected of havingone or more of the following: a chronic neurodegenerative conditionwherein mitochondrial fusion, fitness, or trafficking are impaired; adisease or disorder associated with MFN1 or MFN2 dysfunction; a diseaseassociated with mitochondrial fragmentation, dysfunction, ordysmotility; a degenerative neuromuscular condition (such asCharcot-Marie-Tooth disease, ALS, Huntington's disease, Alzheimer'sdisease, Parkinson's disease); hereditary motor and sensory neuropathy,autism, ADOA, muscular dystrophy, Lou Gehrig's disease, cancer,mitochondrial myopathy, DAD, LHON, Leigh syndrome, subacute sclerosingencephalopathy, NARP, MNGIE, MERRF, MELAS, mtDNA depletion, MNGIE,dysautonomic mitochondrial myopathy, mitochondrial Channelopathy,PDCD/PDH, diabetic neuropathy, chemotherapy-induced peripheralneuropathy, crush injury, SCI, TBI, stroke, optic nerve injury, and/orrelated conditions that involve axonal disconnection.

In some aspects, a method of screening one or more candidate moleculesfor mitochondrial fusion modulatory activity may comprise one or more ofthe following: (i) constitutively expressing a mitochondrial-targetedphotoswitchable fluorophore in cells expressing different combinationsof MFN1 or MFN2 in a genetically-defined manner; (ii) photoswitchingmitochondrial-targeted fluorophores in a micro-matrix pattern in cellstransiently or constitutively expressing a mitochondrial-targetedphotoswitchable fluorophore; and (iii) measuring merged/overlayfluorescence in photoswitched mitochondria.

In some aspects, a method of screening one or more candidate moleculesfor mitochondrial fusion modulatory activity may further comprisecomparing the merged/overlay fluorescence of the test mixture with themerged/overlay fluorescence of the control mixture, wherein when themerged/overlay fluorescence of the test mixture is greater than themerged/overlay fluorescence of the control mixture, the one or morecandidate molecules in the test mixtures is identified as an activatorof mitochondrial fusion.

In some aspects, a method of screening one or more candidate moleculesfor mitochondrial fusion modulatory activity may further comprisecomparing the merged/overlay fluorescence of the test mixture of acandidate agent in wild-type, MFN1, or MFN2 expressing cells with themerged/overlay fluorescence of that candidate agent in cells lackingboth MFN1 and MFN2 (MFN null cells), wherein the merged/overlayfluorescence of the mixture in MFN expressing cells is greater than themerged/overlay fluorescence of the mixture in MFN null cells, the one ormore candidate molecules in the test mixtures is identified as amitofusin activator.

The terms “imine” or “imino,” as used herein, unless otherwiseindicated, include a functional group or chemical compound containing acarbon-nitrogen double bond. The expression “imino compound,” as usedherein, unless otherwise indicated, refers to a compound that includesan “imine” or an “imino” group as defined herein. The “imine” or “imino”group may be optionally substituted.

The term “hydroxy,” as used herein, unless otherwise indicated, includes—OH. The “hydroxy” may be optionally substituted (e.g., incorporated inan alkoxide, phenoxide, or carboxylic acid ester).

The terms “halogen” and “halo”, as used herein, unless otherwiseindicated, include a chlorine, chloro, CI; fluorine, fluoro, F; bromine,bromo, Br; and iodine, iodo, or I.

The term “acetamide,” as used herein, is an organic compound with theformula CH₃CONH₂. The “acetamide” may be optionally substituted.

The term “aryl,” as used herein, unless otherwise indicated, includes acarbocyclic aromatic group. Examples of aryl groups include, but are notlimited to, phenyl, benzyl, naphthyl, and anthracenyl. The “aryl” may beoptionally substituted.

The terms “amine” and “amino”, as used herein, unless otherwiseindicated, include a functional group that contains a nitrogen atom witha lone pair of electrons and wherein one or more hydrogen atoms havebeen replaced by a substituent such as, but not limited to, an alkylgroup or an aryl group. The “amine” or “amino” group may be optionallysubstituted.

The term “alkyl,” as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight or branchedmoieties, such as but not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, and octyl groups. Representative straight-chain loweralkyl groups include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl. Branched lower alkylgroups include, but are not limited to, isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 1-hexyl,2-hexyl, 3-hexyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl,2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl,2-methylheptyl, and 3-methylheptyl. Unsaturated alkyl groups may bereferred to as alkenyl (at least one carbon-carbon double bond) oralkynyl (at least one carbon-carbon triple bond) groups, which mayinclude, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, or 3-methyl-1 butynyl. Eand Z isomers may be present in any alkenyl group. The “alkyl,”“alkenyl,” or “alkynyl” may be optionally substituted.

The term “carboxyl,” as used herein, unless otherwise indicated,includes a functional group containing a carbon atom double bonded to anoxygen atom and single bonded to a hydroxyl group (—COOH). The“carboxyl” may be optionally substituted.

The term “acyl,” as used herein, unless otherwise indicated, includes afunctional group derived from an aliphatic carboxylic acid by removal ofthe hydroxy (—OH) group. The “acyl” may be optionally substituted.

The term “alkoxy,” as used herein, unless otherwise indicated, includesO-alkyl groups wherein alkyl is as defined above and O representsoxygen. Representative alkoxy groups include, but are not limited to,—O-methyl, —O-ethyl, —O-n-propyl, —O-n-butyl, —O-n-pentyl, —O-n-hexyl,—O-n-heptyl, —O-n-octyl, —O-isopropyl, —O-sec-butyl, —O-isobutyl,—O-tert-butyl, —O-isopentyl, —O-2-methylbutyl, —O-2-methylpentyl,—O-3-methylpentyl, —O-2,2-dimethylbutyl, —O-2,3-dimethylbutyl,—O-2,2-dimethylpentyl, —O-2,3-dimethylpentyl, —O-3,3-dimethylpentyl,—O-2,3,4-trimethylpentyl, —O-3-methylhexyl, —O-2,2-dimethylhexyl,—O-2,4-dimethylhexyl, —O-2,5-dimethylhexyl, —O-3,5-dimethylhexyl,—O-2,4-dimethylpentyl, —O-2-methylheptyl, —O-3-methylheptyl, —O-vinyl,—O-allyl, —O-1-butenyl, —O-2-butenyl, —O— isobutylenyl, —O-1-pentenyl,—O-2-pentenyl, —O-3-methyl-1-butenyl, —O-2-methyl-2-butenyl,—O-2,3-dimethyl-2-butenyl, —O-1-hexyl, —O-2-hexyl, —O-3-hexyl,—O-acetylenyl, —O-propynyl, —O-1-butynyl, —O-2-butynyl, —O-1-pentynyl,—O-2-pentynyl, —O-3-methyl-1-butynyl, —O— cyclopropyl, —O-cyclobutyl,—O-cyclopentyl, —O-cyclohexyl, —O-cycloheptyl, —O-cyclooctyl,—O-cyclononyl, —O-cyclodecyl, —O—CH₂-cyclopropyl, —O—CH₂-cyclobutyl,—O—CH₂-cyclopentyl, —O—CH₂-cyclohexyl, —O—CH₂-cycloheptyl,—O—CH₂-cyclooctyl, —O—CH₂-cyclononyl, —O—CH₂-cyclodecyl,—O—(CH₂)_(n)-cyclopropyl, —O—(CH₂)_(n)-cyclobutyl,—O—(CH₂)_(n)-cyclopentyl, —O—(CH₂)_(n)-cyclohexyl,—O—(CH₂)_(n)-cycloheptyl, —O—(CH₂)_(n)-cyclooctyl,—O—(CH₂)_(n)-cyclononyl, and/or —O—(CH₂)_(n)-cyclodecyl. The alkoxy maybe saturated, partially saturated, or unsaturated. The “alkoxy” may beoptionally substituted. In any example above, n may be from one to abouttwenty.

The term “cycloalkyl,” as used herein, unless otherwise indicated,includes a non-aromatic, saturated, partially saturated, or unsaturated,monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbonreferred to herein containing a total of from 3 to 10 carbon atoms.Examples of cycloalkyls include, but are not limited to, C₃₋₁₀cycloalkyl groups including cyclopropyl, cyclobutyl, cyclopentyl,cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl,1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. The term“cycloalkyl” also includes lower alkyl-cycloalkyl, wherein lower alkyland cycloalkyl are as defined herein. Examples of lower alkyl-cycloalkylgroups include, but are not limited to, —CH₂-cyclopropyl,—CH₂-cyclobutyl, —CH₂-cyclopentyl, —CH₂-cyclopentadienyl,—CH₂-cyclohexyl, —CH₂-cycloheptyl, and/or —CH₂-cyclooctyl. The“cycloalkyl” may be optionally substituted.

The term “heterocyclic”, as used herein, unless otherwise indicated,includes an aromatic group or a non-aromatic cycloalkyl group in whichone to four of the ring carbon atoms are independently replaced with oneor more of O, S, and N. Aromatic heterocyclic groups are referred to as“heteroaryl” groups. Non-aromatic heterocyclic groups are referred to as“heterocyclyl” groups. Representative examples of heterocyclic groupsinclude, but are not limited to, benzofuranyl, benzothiophene, indolyl,benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl,thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl,quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl,isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane,4,5-dihydro-1H-imidazolyl, and/or tetrazolyl. Heterocyclic groups may besubstituted or unsubstituted. Heterocyclic groups may also be bonded atany ring atom (i.e., at any carbon atom or heteroatom of theheterocyclic ring). The heterocyclic group may be saturated, partiallysaturated, or unsaturated.

The term “indole,” as used herein, is an aromatic heterocyclic organiccompound with formula C₈H₇N. It has a bicyclic structure containing asix-membered benzene ring fused to a five-membered nitrogen-containingpyrrole ring. The “indole” may be optionally substituted.

The term “cyano,” as used herein, unless otherwise indicated, includes aCN group.

The term “alcohol,” as used herein, unless otherwise indicated, includesa compound in which a hydroxy functional group (—OH) is bound to acarbon atom. In particular, this carbon atom may be saturated, havingsingle bonds to three other atoms. The “alcohol” may be optionallysubstituted. The “alcohol” may be a primary, secondary, or tertiaryalcohol.

The term “solvate” is intended to mean a solvated form of a specifiedcompound that retains the effectiveness of such compound. Examples ofsolvates include compounds of the invention in combination with, but notlimited to, one or more of: water, isopropanol, ethanol, methanol,dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.

The term “mmol,” as used herein, is intended to mean millimole. The term“equiv” and “eq.,” as used herein, are intended to mean equivalent. Theterm “mL,” as used herein, is intended to mean milliliter. The term “g,”as used herein, is intended to mean gram. The term “kg,” as used herein,is intended to mean kilogram. The term “μg,” as used herein, is intendedto mean micrograms. The term “h,” as used herein, is intended to meanhour. The term “min,” as used herein, is intended to mean minute. Theterm “M,” as used herein, is intended to mean molar. The term “μL,” asused herein, is intended to mean microliter. The term “μM,” as usedherein, is intended to mean micromolar. The term “nM,” as used herein,is intended to mean nanomolar. The term “N,” as used herein, is intendedto mean normal. The term “amu,” as used herein, is intended to meanatomic mass unit. The term “° C.,” as used herein, is intended to meandegree Celsius. The term “wt/wt,” as used herein, is intended to meanweight/weight. The term “v/v,” as used herein, is intended to meanvolume/volume. The term “MS,” as used herein, is intended to mean massspectrometry. The term “HPLC,” as used herein, is intended to mean highperformance liquid chromatography. The term “RT,” as used herein, isintended to mean room temperature or retention time, depending oncontext. The term “e.g.,” as used herein, is intended to mean forexample. The term “N/A,” as used herein, is intended to mean not testedor not applicable.

As used herein, the expression “pharmaceutically acceptable salt” refersto pharmaceutically acceptable organic or inorganic salts of a compoundof the invention. Suitable salts include, but are not limited, tosulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and/or pamoate 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion, or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt may have multiple counterions. Hence, a pharmaceuticallyacceptable salt may have one or more charged atoms and/or one or morecounterion. As used herein, the expression “pharmaceutically acceptablesolvate” refers to an association of one or more solvent molecules and acompound of the invention. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and/or ethanolamine. As used herein, the expression “pharmaceuticallyacceptable hydrate” refers to a compound of the invention, or a saltthereof, that further includes a stoichiometric or non-stoichiometricamount of water bound by non-covalent intermolecular forces.

Each of the states, diseases, disorders, and conditions, describedherein, as well as others, can benefit from compositions and methodsdescribed herein. Generally, treating a state, disease, disorder, orcondition includes preventing or delaying the appearance of clinicalsymptoms in a mammal that may be afflicted with or predisposed to thestate, disease, disorder, or condition but does not yet experience ordisplay clinical or subclinical symptoms thereof. Treating can alsoinclude inhibiting the state, disease, disorder, or condition, e.g.,arresting or reducing the development of the disease or at least oneclinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Mitofusin 1 and Mitofusin 2

Mitochondria generate adenosine triphosphate (ATP) that fuels neuronalactivity. Mitochondria must fuse in order to exchange genomes andpromote mutual repair. The initial stages of mitochondrial fusionproceed through the physiochemical actions of two functionally redundantand structurally related dynamin family GTPases, MFN1 and MFN 2. Theobligatory first step leading to mitochondrial fusion is moleculartethering of two mitochondria via homo- or hetero-oligomerization (intrans) of extended MFN1 or MFN2 carboxyl termini. Subsequently, GTPbinding to and hydrolysis by MFN1 or MFN2 promotes irreversible physicalfusion of the organellar outer membranes.

Mitofusins belong to a class of highly conserved GTPases that arelocated on the outer membrane of mitochondria in mammals, flies, theworm, and budding yeast. Each of MFN1 and MFN2, the mitofusins presentin mammals, are anchored to the outer membrane by two transmembranedomains such that their N-terminus and C-terminus are exposed to thecytoplasm. Mitofusins on different organelles undergo transdimerizationthrough anti-parallel binding of their extended carboxy terminalα-helical domains to form mitochondria-mitochondria tethers—the obligateinitial step in mitochondrial fusion (Koshiba et al., 2004, Science,305:858-861). Conventional wisdom is that mitofusins existconstitutively in this “active” extended molecular conformation, whichsupports mitochondrial tethering, although other possible conformationsand the likelihood of functionally relevant molecular plasticity havenot been rigorously tested. The components involved in mitochondrialtethering involve intermolecular and possibly intramolecularinteractions of particular MFN1 and MFN2 domains. These interactionswere further studied and exploited in the design and testing ofcompositions which affect the interactions and the resultantmitochondrial function.

MFN1 and MFN2 share a common domain structure. The amino terminalglobular GTPase domain is followed by a coiled-coiled heptad repeatregion (HR1), two adjacent small transmembrane domains, and a carboxylterminal coiled heptad repeat region (HR2). Amino acid conservationbetween MFN1 and MFN2 varies by domain, being most highly conserved inthe GTPase, transmembrane, and HR2 domains. HR2 domains extending fromMFN1 molecules located on different mitochondria may bind to each other,forming inter-molecular HR2-HR2 interactions that link the molecules andtether the organelles (Koshiba et al., ibid). HR2 may also bind to HR1(Huang et al., 2011, PLoS One, 6:e20655; Franco et al Nature 2016.),

The crystal structure of bacterial dynamin-like protein (DLP) (Low andLowe, 2006, Nature, 444:766-769; Protein Data Bank (PDB) ID No. 2J69)was used to model MFN2 structure. The alignment and modeling of MFN2based on the DLP structure provided a template for the expansion andrefining of the identities of HR2 amino acids that mediateinter-molecular HR2-HR2 tethering (Koshiba et al., 2004, Science,305:858-861). This analysis led to the conception that these same aminoacids mediate, via peptide-peptide interactions, intra-molecularantiparallel binding of HR2 to HR1.

Mitochondria-Associated Diseases, Disorders, or Conditions

The present disclosure provides for compositions and methods oftreatment for treating mitochondria-related diseases, disorders, orconditions, including diseases or disorders associated with MFN1 and/orMFN2 and mitochondrial dysfunction. A mitochondria-associated disease,disorder, or condition may be a disease primarily caused by orsecondarily associated with mitochondrial dysfunction, fragmentation, orloss-of-fusion, or associated with dysfunction in MFN1 or MFN2 catalyticactivity or conformational unfolding. Mitochondrial dysfunction may becaused by genetic mutations of mitofusins or other (nuclear ormitochondrial encoded) genes, or may be caused by physical, chemical, orenvironmental injury to the CNS or PNS.

Mitochondria transit within cells and undergo fusion to exchange genomesand promote mutual repair. Mitochondrial fusion and subcellulartrafficking are mediated in part by MFN1 and MFN2. Genetic mutations inMFN2 that suppress mitochondrial fusion and motility causeCharcot-Marie-Tooth Disease, type 2A (CMT2A), the most common heritableaxonal neuropathy. Mitochondrial fragmentation, dysfunction, anddysmotility are also central features of other genetic neurodegenerativesyndromes, such as amyotrophic lateral sclerosis, Huntington's disease,Parkinson's disease, and Alzheimer's disease. Because no therapeuticsexist that directly enhance mitochondrial fusion or trafficking, thesediseases are unrelenting and considered irreversible.

Examples of mitochondria-associated diseases, disorders, and conditionsinclude, but are not limited to, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Charcot-Marie-Tooth Disease (type 2A)(CMT), hereditary motor and sensory neuropathy, autism, ADOA, musculardystrophy, Lou Gehrig's disease, cancer, mitochondrial myopathy, DAD,LHON, Leigh syndrome, subacute sclerosing encephalopathy, NARP, MNGIE,MERRF, MELAS, mtDNA depletion, MNGIE, dysautonomic mitochondrialmyopathy, mitochondrial channelopathy, and/or PDCD/PDH.

Symptoms that may be treated with the methods as described hereininclude, but are not limited to, poor growth, loss of musclecoordination, muscle paralysis and atrophy, visual problems, hearingproblems, learning disabilities, heart disease, liver disease, kidneydisease, gastrointestinal disorders, respiratory disorders, neurologicalproblems, autonomic dysfunction, and dementia.

Neurodegenerative Disease

As described herein, mitofusin activators rapidly reverse mitochondrialdysmotility in sciatic nerve axons of a mouse model ofCharcot-Marie-Tooth disease, type 2A. Because impaired mitochondrialfusion, fitness, and/or trafficking also contribute to neuronaldegeneration in various neurodegenerative diseases (e.g., inCharcot-Marie-Tooth disease (CMT2A), Huntington's disease, Parkinson'sdisease, and Alzheimer's disease, and especially in ALS), the presentdisclosure provides for compositions (e.g., compositions containingmitofusin activators) and methods to treat such neurodegenerativediseases, disorders, and/or conditions.

Examples of neurodegenerative diseases, disorders and conditions includea disease of impaired neuronal mitochondrial dynamism or trafficking,such as, but not limited to, a hereditary motor and sensory neuropathy(HMSN) (e.g., CMT1 (a dominantly inherited, hypertrophic, predominantlydemyelinating form), CMT2 (a dominantly inherited predominantly axonalform), Dejerine-Sottas (severe form with onset in infancy), CMTX(inherited in an X-linked manner), and CMT4 (includes the variousdemyelinating autosomal recessive forms of Charcot-Marie-Tooth disease);hereditary sensory and autonomic neuropathy type IE, hereditary sensoryand autonomic neuropathy type II, hereditary sensory and autonomicneuropathy type V, HMSN types 1A and 1B (e.g., dominantly inheritedhypertrophic demyelinating neuropathies), HMSN type 2 (e.g., dominantlyinherited neuronal neuropathies), HMSN type 3 (e.g., hypertrophicneuropathy of infancy [Dejerine-Sottas]), HMSN type 4 (e.g.,hypertrophic neuropathy [Refsum] associated with phytanic acid excess),HMSN type 5 (associated with spastic paraplegia), and/or HMSN type 6(e.g., with optic atrophy)).

Other examples of neurodegenerative diseases, disorders, and conditionsinclude, but are not limited to, Alzheimer's disease, ALS, Alexanderdisease, Alpers' disease, Alpers-Huttenlocher syndrome,alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Artssyndrome, ataxia neuropathy spectrum, ataxia (e.g., with oculomotorapraxia, autosomal dominant cerebellar ataxia, deafness, andnarcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay,Batten disease, beta-propeller protein-associated neurodegeneration,cerebro-oculo-facio-skeletal syndrome (COFS), corticobasal degeneration,CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease,CLN6 disease, CLN7 disease, CLN8 disease, cognitive dysfunction,congenital insensitivity to pain with anhidrosis, dementia, familialencephalopathy with neuroserpin inclusion bodies, familial Britishdementia, familial Danish dementia, fatty acid hydroxylase-associatedneurodegeneration, Friedreich's Ataxia, Gerstmann-Straussler-ScheinkerDisease, GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., withretinitis pigmentosa), Huntington's disease, infantile neuroaxonaldystrophy, infantile-onset ascending hereditary spastic paralysis,infantile-onset spinocerebellar ataxia, juvenile primary lateralsclerosis, Kennedys disease, Kuru, Leigh's Disease, Marinesco-Sjogrensyndrome, mild cognitive impairment (MCI), mitochondrial membraneprotein-associated neurodegeneration, motor neuron disease, monomelicamyotrophy, motor neuron diseases (MND), multiple system atrophy,multiple system atrophy with orthostatic hypotension (Shy-DragerSyndrome), multiple sclerosis, multiple system atrophy,neurodegeneration in down's syndrome (NDS), neurodegeneration of aging,neurodegeneration with brain iron accumulation, neuromyelitis optica,pantothenate kinase-associated neurodegeneration, opsoclonus myoclonus,prion disease, progressive multifocal leukoencephalopathy, Parkinson'sdisease, Parkinson's disease-related disorders, polycysticlipomembranous osteodysplasia with sclerosing leukoencephalopathy, priondisease, progressive external ophthalmoplegia, riboflavin transporterdeficiency neuronopathy, Sandhoff disease, spinal muscular atrophy(SMA), spinocerebellar ataxia (SCA), striatonigral degeneration,transmissible spongiform encephalopathies (prion diseases), and/orWallerian-like degeneration.

CHARCOT-MARIE-TOOTH (CMT) DISEASE TYPE 2A.

Charcot-Marie-Tooth type 2A (CMT2A) disease is an example of anon-curable neurodegenerative disease/axonal neuropathy, disorder, orcondition caused by mutations of MFN2 and for which there are currentlyno disease-modifying treatments. As described herein, it was discoveredthat severely impaired mitochondrial transport from neuron cell body inthe spinal cord to distal neuronal synapse in the lower leg or hand (inaddition to smaller mitochondria size as is widely recognized) is acentral factor in CMT2A disease onset and progression. CMT2A is aprogressive neuromuscular disease that typically causes muscle weaknessand wasting in the distal legs/feet in children of ages 1-8 years, thenupper limbs, ultimately producing severe muscle wasting, skeletaldeformities, and permanent disability. The present disclosure providesfor the correction of impaired neuronal mitochondria transport as atherapeutic target in this disease. Data showed that administration of amitofusin activator promoted the mitochondria to move along neuronalaxons in mouse models where mitochondria were not previously moving,which is applicable in any neuropathy (e.g., Huntington's disease, ALS,ALS-like sclerosis, and/or Alzheimer's disease).

Neurological Disease as Described in Franco et al. Nature 2016 and Rochaet al. Science 2018

As described herein, mitofusin activators rapidly reverse mitochondrialdysmotility in sciatic nerve axons of a mouse model ofCharcot-Marie-Tooth disease type 2A. It is currently believed thatimpaired mitochondrial trafficking also contribute to neuronaldegeneration in various neurological diseases (e.g., in Huntington'sdisease, Parkinson's disease, and Alzheimer's disease, and especially inALS). As such, the present disclosure provides for methods andcompositions to treat neurological diseases, disorders, or conditions.For example, a neurological disease, disorder, or condition may be, butis not limited to, abulia; agraphia; alcoholism; alexia; alien handsyndrome; Allan-Herndon-Dudley syndrome; alternating hemiplegia ofchildhood; Alzheimer's disease; amaurosis fugax; amnesia; ALS; aneurysm;angelman syndrome; anosognosia; aphasia; apraxia; arachnoiditis;Arnold-Chiari malformation; asomatognosia; Asperger syndrome; ataxia;attention deficit hyperactivity disorder; atr-16 syndrome; auditoryprocessing disorder; autism spectrum; Behcets disease; bipolar disorder;Bell's palsy; brachial plexus injury; brain damage; brain injury; braintumor; Brody myopathy; Canavan disease; capgras delusion; carpal tunnelsyndrome; causalgia; central pain syndrome; central pontinemyelinolysis; centronuclear myopathy; cephalic disorder; cerebralaneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); cerebraldysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK syndrome);cerebral gigantism; cerebral palsy; cerebral vasculitis; cervical spinalstenosis; Charcot-Marie-Tooth disease; chiari malformation; chorea;chronic fatigue syndrome; chronic inflammatory demyelinatingpolyneuropathy (CIDP); chronic pain; Cockayne syndrome; Coffin-Lowrysyndrome; coma; complex regional pain syndrome; compression neuropathy;congenital facial diplegia; corticobasal degeneration; cranialarteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulativetrauma disorders; Cushing's syndrome; cyclothymic disorder; cyclicvomiting syndrome (CVS); cytomegalic inclusion body disease (CIBD);cytomegalovirus infection; Dandy-Walker syndrome; Dawson disease; deMorsier's syndrome; Dejerine-Klumpke palsy; Dejerine-Sottas disease;delayed sleep phase syndrome; dementia; dermatomyositis; developmentalcoordination disorder; diabetic neuropathy; diffuse sclerosis; diplopia;disorders of consciousness; down syndrome; Dravet syndrome; duchennemuscular dystrophy; dysarthria; dysautonomia; dyscalculia; dysgraphia;dyskinesia; dyslexia; dystonia; empty sella syndrome; encephalitis;encephalocele; encephalotrigeminal angiomatosis; encopresis; enuresis;epilepsy; epilepsy-intellectual disability in females; erb's palsy;erythromelalgia; essential tremor; exploding head syndrome; Fabry'sdisease; Fahr's syndrome; fainting; familial spastic paralysis; febrileseizures; Fisher syndrome; Friedreich's ataxia; fibromyalgia; Foville'ssyndrome; fetal alcohol syndrome; fragile x syndrome; fragilex-associated tremor/ataxia syndrome (FXTAS); Gaucher's disease;generalized epilepsy with febrile seizures plus; Gerstmann's syndrome;giant cell arteritis; giant cell inclusion disease; globoid cellleukodystrophy; gray matter heterotopia; Guillain-Barré syndrome;generalized anxiety disorder; HTLV-1 associated myelopathy;Hallervorden-Spatz syndrome; head injury; headache; hemifacial spasm;hereditary spastic paraplegia; heredopathia atactica polyneuritiformis;herpes zoster oticus; herpes zoster; Hirayama syndrome; Hirschsprung'sdisease; Holmes-Adie syndrome; holoprosencephaly; Huntington's disease;hydranencephaly; hydrocephalus; hypercortisolism; hypoxia;immune-mediated encephalomyelitis; inclusion body myositis;incontinentia pigmenti; infantile refsum disease; infantile spasms;inflammatory myopathy; intracranial cyst; intracranial hypertension;isodicentric 15; Joubert syndrome; Karak syndrome; Kearns-Sayresyndrome; Kinsbourne syndrome; Kleine-Levin syndrome; Klippel Feilsyndrome; Krabbe disease; Kufor-Rakeb syndrome; Lafora disease;Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateralmedullary (Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy;leukoencephalopathy with vanishing white matter; lewy body dementia;lissencephaly; locked-in syndrome; Lou Gehrig's disease (amyotrophiclateral sclerosis (ALS)); lumbar disc disease; lumbar spinal stenosis;lyme disease—neurological sequelae; Machado-Joseph disease(spinocerebellar ataxia type 3); macrencephaly; macropsia; mal dedebarquement; megalencephalic leukoencephalopathy with subcorticalcysts; megalencephaly; Melkersson-Rosenthal syndrome; menieres disease;meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly;micropsia; migraine; Miller Fisher syndrome; mini-stroke (transientischemic attack); misophonia; mitochondrial myopathy; mobius syndrome;monomelic amyotrophy; Morvan syndrome; motor neurone disease—see ALS;motor skills disorder; moyamoya disease; mucopolysaccharidoses;multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis;multiple system atrophy; muscular dystrophy; myalgic encephalomyelitis;myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonicencephalopathy of infants; myoclonus; myopathy; myotubular myopathy;myotonia congenita; narcolepsy; neuro-Behcet's disease;neurofibromatosis; neuroleptic malignant syndrome; neurologicalmanifestations of aids; neurological sequelae of lupus; neuromyotonia;neuronal ceroid lipofuscinosis; neuronal migration disorders;neuropathy; neurosis; Niemann-Pick disease; non-24-hour sleep-wakedisorder; nonverbal learning disorder; O'Sullivan-McLeod syndrome;occipital neuralgia; occult spinal dysraphism sequence; Ohtaharasyndrome; olivopontocerebellar atrophy; opsoclonus myoclonus syndrome;optic neuritis; orthostatic hypotension; otosclerosis; overuse syndrome;palinopsia; paresthesia; Parkinson's disease; paramyotonia congenita;paraneoplastic diseases; paroxysmal attacks; Parry-Romberg syndrome;pediatric autoimmune neuropsychiatric disorders associated withstreptococcoal infections (PANDAS); Pelizaeus-Merzbacher disease;periodic paralyses; peripheral neuropathy; pervasive developmentaldisorders; phantom limb/phantom pain; photic sneeze reflex; phytanicacid storage disease; Pick's disease; pinched nerve; pituitary tumors;pmg; polyneuropathy; polio; polymicrogyria; polymyositis; porencephaly;post-polio syndrome; postherpetic neuralgia (phn); postural hypotension;Prader-Willi syndrome; primary lateral sclerosis; prion diseases;progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive supranuclear palsy; prosopagnosia;pseudotumor cerebri; quadrantanopia; quadriplegia; rabies;radiculopathy; Ramsay Hunt syndrome type 1; Ramsay Hunt syndrome type 2;Ramsay Hunt syndrome type 3—see Ramsay-Hunt syndrome; Rasmussenencephalitis; reflex neurovascular dystrophy; refsum disease; REM sleepbehavior disorder; repetitive stress injury; restless legs syndrome;retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome;rhythmic movement disorder; Romberg syndrome; Saint Vitus' dance;Sandhoff disease; Schilder's disease (two distinct conditions);schizencephaly; sensory processing disorder; septo-optic dysplasia;shaken baby syndrome; shingles; Shy-Drager syndrome; Sjogren's syndrome;sleep apnea; sleeping sickness; snatiation; Sotos syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; spinal and bulbar muscular atrophy; spinocerebellar ataxia;split-brain; Steele-Richardson-Olszewski syndrome; stiff-personsyndrome; stroke; Sturge-Weber syndrome; stuttering; subacute sclerosingpanencephalitis; subcortical arteriosclerotic encephalopathy;superficial siderosis; Sydenham's chorea; syncope; synesthesia;syringomyelia; tarsal tunnel syndrome; tardive dyskinesia; tardivedysphrenia; Tarlov cyst; Tay-Sachs disease; temporal arteritis; temporallobe epilepsy; tetanus; tethered spinal cord syndrome; Thomsen disease;thoracic outlet syndrome; tic douloureux; Todd's Paralysis; tourettesyndrome; toxic encephalopathy; transient ischemic attack; transmissiblespongiform encephalopathies; transverse myelitis; traumatic braininjury; tremor; trichotillomania; trigeminal neuralgia; tropical spasticparaparesis; trypanosomiasis; tuberous sclerosis; 22q13 deletionsyndrome; Unverricht-Lundborg disease; vestibular schwannoma (acousticneuroma); Von Hippel-Lindau disease (VHL); viliuisk encephalomyelitis(VE); Wallenberg's syndrome; west syndrome; whiplash; Williams syndrome;Wilson's disease; y-linked hearing impairment; and/or Zellwegersyndrome.

Cns or Pns Injury or Trauma

Injury in the CNS or PNS (e.g., trauma to the CNS or PNS, crush injury,SCI, TBI, stroke, optic nerve injury, or related conditions that involveaxonal disconnection) may be treated with the compositions and methodsas described herein. The CNS includes the brain and the spinal cord andthe PNS is composed of cranial, spinal, and autonomic nerves thatconnect to the CNS.

Damage to the nervous system caused by mechanical, thermal, chemical, orischemic factors may impair various nervous system functions such asmemory, cognition, language, and voluntary movement. Most often, this isthrough accidental crush or transection of nerve tracts, or as anunintended consequence of medical therapy for cancer using chemotherapy.This results in the interruption of communication between nerve cellbodies and their targets. Other types of injuries may include disruptionof the interrelations between neurons and their supporting cells or thedestruction of the blood-brain barrier.

As described herein, mitofusin activators rapidly reverse mitochondrialdysmotility in neurons from mice with various genetic neurodegenerativediseases and in axons injured or severed by physical injury. For thisreason, it is believed that enhancing mitochondrial trafficking withmitofusin activators may enhance regeneration/repair of physicallydamaged nerves, as in vehicular and sports injuries, penetration traumafrom military or criminal actions, and inatrogenic injury duringinvasive medical procedures. Further testing of the injury-regenerationhypothesis will be further developed with the small molecule mitofusinactivators for evaluation of their in vivo effectiveness. As such, thepresent disclosure provides for compositions and methods to treatphysical nerve injury.

As disclosed herein, mitochondria motility was implicated in neuropathy.It is believed that mitochondrial motility is also implicated in nerveinjuries, especially in nerves that have not severed, such as a crushinjury. After an accident or crush injury, nerves will regenerate ordie. Small molecule mitofusin activators, as described herein, mayincrease mitochondrial trafficking, enabling the nerve to regenerateafter a crush injury.

Formulation

The agents and compositions described herein may be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described previously (e.g., Remington'sPharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), which is incorporated herein by reference withrespect to its disclosure of pharmaceutically acceptable carriers). Suchformulations will contain a therapeutically effective amount of abiologically active agent described herein, which may be in purifiedform, together with a suitable amount of carrier to provide the form forproper administration to a subject.

The term “formulation” refers to a preparation of a drug in a formsuitable for administration to a subject such as a human. Thus, a“formulation” may include pharmaceutically acceptable excipients,including diluents or carriers.

The term “pharmaceutically acceptable,” as used herein, describessubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. One ofskill in the art will be familiar with suitable pharmaceuticallyacceptable substances. Examples of pharmaceutically acceptableingredients include those having monographs in United StatesPharmacopeia (USP 29) and National Formulary (NF 24), United StatesPharmacopeial Convention, Inc, Rockville, Md., 2005 (“USP/NF”), or amore recent edition, and the components listed in the continuouslyupdated Inactive Ingredient Search online database of the FDA. Otheruseful components that are not described in the USP/NF may also be used.

The term “pharmaceutically acceptable excipient,” as used herein,includes solvents, dispersion media, coatings, antibacterial agents,antifungal agents, isotonic, and absorption delaying agents. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art (see generally Remington's Pharmaceutical Sciences (A.R.Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar asany conventional media or agent is incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients may also be incorporated into thecompositions.

A “stable” formulation or composition refers to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

A formulation should suit the desired mode of administration. The agentsof use with the current disclosure may be formulated by known methodsfor administration to a subject using several routes including, but notlimited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations may also be used to affect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period. In order tomaintain a near-constant level of an agent in the body, the agent may bereleased from the dosage form at a rate that will replace the amount ofagent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers(e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules).

Agents or compositions described herein may also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Also provided herein is a process of treating a mitochondria-associateddisease, disorder, or condition in a subject in need of administrationof a therapeutically effective amount of mitofusin activator to preventor treat a mitochondria-associated disease, disorder, or condition.

For example, the compositions and methods described herein may be usedas a primary therapy for Charcot-Marie-Tooth or as an adjunctive therapyfor Huntington's disease, Parkinson's disease, Alzheimer's disease, orALS to retard or reverse disease progression.

As another example, the compositions and methods described herein may beused for the treatment of a physical injury. For example, as a primarytherapy for any contusive injury involving the spine or peripheralnerves (perhaps even the brain, i.e., concussion), such as motor vehicleor sports injuries. This therapy may help restore normal motor functionby augmenting regeneration and repair of injured neurons.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the therapeutic methods described hereinmay be a subject having, diagnosed with, suspected of having, or at riskfor developing a mitochondria-associated disease, disorder, orcondition. A determination of the need for treatment will typically beassessed by a history and physical exam consistent with the disease orcondition at issue. Diagnosis of the various conditions treatable by themethods described herein is within the skill of the art. The subject maybe an animal subject, including a mammal, such as horses, cows, dogs,cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, chickens,and humans. For example, the subject may be a human subject.

Generally, a safe and effective amount of a mitofusin activator is, forexample, that amount that would cause the desired therapeutic effect ina subject while minimizing undesired side effects. In various aspects,an effective amount of a mitofusin activator described herein maysubstantially inhibit mitochondria-associated disease, disorder, orcondition, slow the progress of mitochondria-associated disease,disorder, or condition, or limit the development ofmitochondria-associated disease, disorder, or condition. For example, adesired therapeutic effect may be a delay in peripheral neuropathy(e.g., over the course of three years) compared to placebo assessed byslower increase in modified composite CMT neuropathy score. As anotherexample, a desired therapeutic effect may be reversal or absence ofprogression of peripheral neuropathy compared to placebo, as indicatedby lower or stable modified composite CMT neuropathy score. As yetanother example, a desired therapeutic effect may be reversal or absenceof progression of dysregulated motor function or increased regenerationand repair of injured neurons.

According to the methods described herein, administration may beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of a mitofusin activator may be employed in pure formor, where such forms exist, in pharmaceutically acceptable salt form andwith or without a pharmaceutically acceptable excipient. For example,the compounds of the present disclosure may be administered, at areasonable benefit/risk ratio applicable to any medical treatment, in asufficient amount to treat, prevent, or slow the progression ofmitochondria-associated disease, disorder, or condition.

The amount of a composition described herein that may be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein maybe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that may be expressed as the ratio LD₅₀/ED₅₀,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Shargel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, may benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingmay also include inhibiting the state, disease, disorder, or condition(e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof). Furthermore, treating mayinclude relieving the disease (e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms). A benefit to a subject to be treated may beeither statistically significant or at least perceptible to the subjector to a physician.

Administration of a mitofusin activator may occur as a single event orover a time course of treatment. For example, a mitofusin activator maybe administered daily, weekly, bi-weekly, or monthly. For treatment ofacute conditions, the time course of treatment will usually be at leastseveral days. Certain conditions could extend treatment from severaldays to several weeks. For example, treatment could extend over oneweek, two weeks, or three weeks. For chronic conditions, treatment couldextend from several weeks to several months or even years.

Treatment in accord with the methods described herein may be performedprior to, concurrent with, or after conventional treatment modalitiesfor treating, preventing, or slowing the progression ofmitochondria-associated disease, disorder, or condition.

A mitofusin activator may be administered simultaneously or sequentiallywith another agent, such as an antibiotic, an anti-inflammatory, oranother neuroregenerative agent. For example, a mitofusin activator maybe administered simultaneously with another agent, such as an antibioticor an anti-inflammatory. Simultaneous administration may occur throughadministration of separate compositions, each containing one or more ofa mitofusin activator, an antibiotic, an anti-inflammatory, or anotheragent. Simultaneous administration may occur through administration ofone composition containing two or more of mitofusin activator, anantibiotic, an anti-inflammatory, or another agent. A mitofusinactivator may be administered sequentially with an antibiotic, ananti-inflammatory, or another agent. For example, a mitofusin activatormay be administered before or after administration of an antibiotic, ananti-inflammatory, or another agent.

Administration

Agents and compositions described herein may be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition may be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

As discussed above, administration may be parenteral, pulmonary, oral,topical, transdermal (e.g., a transdermal patch) intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein may be administered in avariety of methods well known in the arts. Administration methods mayinclude, for example, methods involving oral ingestion, direct injection(e.g., systemic or stereotactic), implantation of cells engineered tosecrete the factor of interest, drug-releasing biomaterials, polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, implantable matrix devices, mini-osmoticpumps, implantable pumps, injectable gels and hydrogels, liposomes,micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm),microspheres (e.g., 1-100 μm), reservoir devices, a combination of anyof the above, or other suitable delivery vehicles to provide the desiredrelease profile in varying proportions. Other methods ofcontrolled-release delivery of agents or compositions will be known tothe skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump that may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition may beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system may be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents may be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to nontarget tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Screening

Also provided are methods for screening. As described herein, an imagingmethod for screening and evaluating small molecular or other regulatorsof mitochondrial fusion is provided.

The term “mitochondrial fusion,” as used herein, refers to the physicalmerging and transfer of components between two or more previouslydistinct mitochondria.

Mitochondrial fusion is distinct from an increase in mitochondrial“aspect ratio” (the ratio of the mitochondrial long/short axis, ormitochondrial length/width) because it is impossible to discriminatebetween increases in mitochondrial aspect ratio that occur due toincreased mitochondrial fusion versus, for example, decreasedmitochondrial fission.

With the mitofusin activators that have been shown to activatemitochondrial fusion, assays may be designed and performed to screencandidate agents or molecules for specific compositions that mayactivate mitochondrial fusion per se. For example, identification ofsmall molecule mitofusin activators provides an alternate modulatingcomposition that may be more efficient to synthesize and use. Candidateagents encompass numerous chemical classes, typically synthetic,semi-synthetic, or naturally-occurring inorganic or organic molecules.Candidate agents include those found in large libraries of synthetic ornatural compounds. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available fromcommercial resources or are readily producible. In some aspects,mitofusin activators or other small molecule activators of mitochondrialfusion identified through these screening assays may become promisingtherapeutic agents for treating diseases or disorders associated withdefects in mitochondrial fusion.

A screening assay for mitochondrial fusion may use amitochondrial-targeted photoswitchable fluorophore geneticallyintroduced into cultured cells expressing any complement of mammalianmitofusins (e.g., both MFN1 and MFN2 [wild-type], MFN1 alone [MFN2null], MFN2 alone [MFN1 null], or neither MFN1 nor MFN2 [MFN1/MFN2double null]). In this assay, cells constitutively expressing thephotoswitchable mitochondrial fluorophore are cultured on microscopeslides or on the well surfaces of a high throughput screen plate.Patterned laser illumination of the cells promotes photo switching in amatrix pattern, which converts half of the mitochondria in each cellfrom green to red fluorescence. Photo switched cells are then incubatedwith vehicle (negative control), mitofusin activators (positivecontrol), or unknown compounds for increasing periods of time (e.g., afraction of an hour, 1 hour, 2 hours, 3 hours, or multiples thereof). Acandidate agent is assessed for its ability to stimulate co-localizationof red and green fluorescence within the same mitochondria, visuallyassessed via microscopy or automated imaging as the presence of yellow(green+red) mitochondria in the same cell. An agent that stimulatesmitochondrial fusion will increase red/green colocalization at a giventime point after treatment. A candidate agent able to promote red/greenmitochondrial colocalization 30%, 50%, or >70% greater than vehicle, orcomparable (within 50% at similar doses) to a validated mitofusinactivator, may activate mitochondrial fusion.

The subject methods find use in the screening of a variety of differentcandidate molecules (e.g., potentially therapeutic candidate molecules).Candidate substances for screening according to the methods describedherein include, but are not limited to, fractions of tissues or cells,nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers,ribozymes, triple helix compounds, antibodies, and small (e.g., lessthan about 1000 Da) organic molecules or inorganic molecules including,but not limited to, salts or metals.

Candidate molecules encompass numerous chemical classes, for example,organic molecules, such as small organic compounds having a molecularweight of more than 50 Da and less than about 2,500 Da. Candidatemolecules may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group, andusually at least two of the functional chemical groups. The candidatemolecules may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

A candidate molecule may be a compound in a library database ofcompounds. One of skill in the art will be generally familiar with, forexample, numerous databases for commercially available compounds forscreening (see e.g., ZINC database, UCSF, with 2.7 million compoundsover 12 distinct subsets of molecules; Irwin and Shoichet (2005) J ChemInf Model 45, 177-182). One of skill in the art will also be familiarwith a variety of search engines to identify commercial sources ordesirable compounds and classes of compounds for further testing (seee.g., ZINC database; eMolecules.com; and electronic libraries ofcommercial compounds provided by vendors, for example: ChemBridge,Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicalsetc.).

Candidate molecules for screening according to the methods describedherein include both lead-like compounds and drug-like compounds. Alead-like compound is generally understood to have a relatively smallerscaffold-like structure (e.g., molecular weight of about 150 to about350 kD) with relatively fewer features (e.g., less than about 3 hydrogendonors and/or less than about 6 hydrogen acceptors; hydrophobicitycharacter xlogP of about −2 to about 4) (see e.g., Angewante (1999)Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compoundis generally understood to have a relatively larger scaffold (e.g.,molecular weight of about 150 KDa to about 500 kDa) with relatively morenumerous features (e.g., less than about ten hydrogen acceptors and/orless than about eight rotatable bonds; hydrophobicity character xlogP ofless than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44,235-249). Initial screening may be performed with lead-like compounds.

When designing a lead from spatial orientation data, it may be useful tounderstand that certain molecular structures are characterized as being“drug-like.” Such characterization may be based on a set of empiricallyrecognized qualities derived by comparing similarities across thebreadth of known drugs within the pharmacopoeia. While it is notrequired for drugs to meet all, or even any, of these characterizations,it is far more likely for a drug candidate to meet with clinical successif it is drug-like.

Several of these “drug-like” characteristics have been summarized intothe four rules of Lipinski (generally known as the “rules of fives”because of the prevalence of the number 5 among them). While these rulesgenerally relate to oral absorption and are used to predictbioavailability of compound during lead optimization, they may serve aseffective guidelines for constructing a lead molecule during rationaldrug design efforts such as may be accomplished by using the methods ofthe present disclosure.

The four “rules of five” state that a candidate drug-like compoundshould have at least three of the following characteristics: (i) aweight less than 500 Da; (ii) a log of P less than 5; (iii) no more thanfive hydrogen bond donors (expressed as the sum of —OH and —NH groups);and (iv) no more than ten hydrogen bond acceptors (the sum of N and Oatoms). In addition, drug-like molecules typically have a span (breadth)of between about 8 Å to about 15 Å.

Fragment-based lead discovery (FBLD) also known as fragment-based drugdiscovery (FBDD) is a method that may be used for finding lead compoundsas part of the drug discovery process. It is based on identifying smallchemical fragments, which may bind only weakly to the biological target,and then growing them or combining them to produce a lead with a higheraffinity. FBLD may be compared with high-throughput screening (HTS). InHTS, libraries with up to millions of compounds, with molecular weightsof around 500 Da, are screened, and nanomolar-binding affinities aresought. In contrast, in the early phase of FBLD, libraries with a fewthousand compounds with molecular weights of around 200 Da may bescreened, and millimolar affinities may be considered useful.

In analogy to the rule of five, it has been proposed that idealfragments could follow the ‘rule of three’ (molecular weight<300 Da,ClogP<3, the number of hydrogen bond donors and acceptors each should beless than three and the number of rotatable bonds should be less thanthree). Since the fragments have relatively low affinity for theirtargets, they should have high water solubility so that they may bescreened at higher concentrations.

In fragment-based drug discovery, the low binding affinities of thefragments may pose significant challenges for screening. Manybiophysical techniques have been applied to address this issue. Inparticular, ligand-observe nuclear magnetic resonance (NMR) methods suchas water-ligand observed via gradient spectroscopy (waterLOGSY),saturation transfer difference spectroscopy (STD-NMR), 19^(F) NMRspectroscopy and inter-ligand Overhauser effect (ILOE) spectroscopy,protein-observe NMR methods such as ¹H/¹⁵N heteronuclear single quantumcoherence (HSQC) that utilizes isotopically-labelled proteins, surfaceplasmon resonance (SPR) and isothermal titration calorimetry (ITC) areroutinely-used for ligand screening and for the quantification offragment binding affinity to the target protein.

Once a fragment (or a combination of fragments) have been identified,protein X-ray crystallography may be used to obtain structural models ofthe protein-fragment(s) complexes. Such information may then be used toguide organic synthesis for high-affinity protein ligands and enzymeinhibitors.

Advantages of screening low molecular weight fragment based librariesover traditional higher molecular weight chemical libraries may include:

-   -   (i) More hydrophilic hits in which hydrogen bonding is more        likely to contribute to affinity (enthalpically driven binding).        It is generally much easier to increase affinity by adding        hydrophobic groups (entropically driven binding); starting with        a hydrophilic ligand increases the chances that the final        optimized ligand will not be too hydrophobic (log P<5).    -   (ii) Higher ligand efficiency so that the final optimized ligand        will more likely be relatively low in molecular weight (MW<500        Da).    -   (iii) Since two to three fragments in theory may be combined to        form an optimized ligand, screening a fragment library of N        compounds is equivalent to screening N2-N3 compounds in a        traditional library.

Fragments may be less likely to contain sterically blocking groups thatinterfere with an otherwise favorable ligand-protein interaction,increasing the combinatorial advantage of a fragment library evenfurther.

Kits

Also provided herein are kits. Such kits may include an agent orcomposition described herein and, in certain aspects, instructions foradministration. Such kits may facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition may be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to MFN1,MFN2, antactivator target peptides, activator target peptides, ormitofusin activators. Such packaging of the components separately can,if desired, be presented in a pack or dispenser device, which maycontain one or more unit dosage forms containing the composition. Thepack may, for example, comprise metal or plastic foil such as a blisterpack. Such packaging of the components separately may also, in certaininstances, permit long-term storage without losing activity of thecomponents.

Kits may also include reagents in separate containers (e.g., sterilewater or saline) to be added to a lyophilized active component packagedseparately. For example, sealed glass ampules may contain a lyophilizedcomponent and in a separate ampule, sterile water, sterile saline orsterile each of which has been packaged under a neutral non-reactinggas, such as nitrogen. Ampules may consist of any suitable material,such as glass, organic polymers, such as polycarbonate, polystyrene,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include bottles that maybe fabricated from similar substances as ampules, and envelopes that mayconsist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, and thelike. Containers may have a sterile access port, such as a bottle havinga stopper that may be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to mix.Removable membranes may be glass, plastic, rubber, and the like.

In certain aspects, kits may be supplied with instructional materials.Instructions may be printed on paper or other substrate, and/or may besupplied as an electronic-readable medium, such as a floppy disc,mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and thelike. Detailed instructions may not be physically associated with thekit; instead, a user may be directed to an Internet web site specifiedby the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols may be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some aspects, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain aspects of the present disclosure areto be understood as being modified in some instances by the term“about.” In some features, the term “about” is used to indicate that avalue includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some features, thenumerical parameters set forth in the written description and attachedclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by a particular feature. In someaspects, the numerical parameters should be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of some aspects of the present disclosureare approximations, the numerical values set forth in the specificexamples are reported as precisely as practicable. The numerical valuespresented in some aspects of the present disclosure may contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. The recitation of ranges of valuesherein is merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein.

In some aspects, the terms “a” and “an” and “the” and similar referencesused in the context of describing a particular aspect (especially in thecontext of certain of the following claims) may be construed to coverboth the singular and the plural, unless specifically noted otherwise.In some aspects, the term “or” as used herein, including the claims, isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” areopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and may cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand may cover other unlisted features.

All methods described herein may be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided with respect to certain aspects herein is intendedmerely to better illuminate the present disclosure and does not pose alimitation on the scope of the present disclosure otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements, embodiments, aspects, or features ofthe present disclosure disclosed herein are not to be construed aslimitations. Each group member may be referred to and claimedindividually or in any combination with other members of the group orother elements found herein. One or more members of a group may beincluded in, or deleted from, a group for reasons of convenience orpatentability. When any such inclusion or deletion occurs, thespecification is herein deemed to contain the group as modified thusfulfilling the written description of all Markush groups used in theappended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments, features, oraspects are possible without departing the scope of the presentdisclosure defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus may be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes may be made in the specific features that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the present disclosure.

Example 1: Identification of Amino Acid Residues in the HR1 MFN1 andMFN2 Domain that Influence Conformation

The following example shows that MFN1 and MFN2 conformation isinfluenced by a plurality of amino acid residues in the first heptadrepeat (HR1) domain.

Mitochondria generate ATP that fuels neuronal activity. Mitochondrialdysfunction is implicated in chronic degenerative neurologicalconditions such as Alzheimer's, Parkinson's, and Huntington's diseases.Mitochondria fuse in order to exchange genomes and promote mutualrepair. The initial stages of mitochondrial fusion proceed through thephysiochemical actions of two closely related dynamin family GTPases,mitofusins (MFN) 1 and 2. The obligatory first step leading tomitochondrial fusion is molecular tethering of two mitochondria viahomo- or hetero-oligomerization (in trans) of extended MFN1 or MFN2carboxyl termini. Subsequently, GTP binding to and hydrolysis by MFN1 orMFN2 promotes irreversible physical fusion of the organellar outermembranes. The genetic neurodegenerative condition, Charcot-Marie-ToothDisease (type 2A) (CMT2A) or hereditary motor and sensory neuropathy, iscaused by any of over 50 loss-of-function mutations of MFN2. Theunderlying mechanism that causes this debilitating neuropathy isimpaired mitochondrial fusion and trafficking due to dominant inhibitionof normal MFN1 and MFN2 by the mutant protein. Currently, there is nodisease-altering treatment for CMT2A.

MFN1 and MFN2 share a common domain structure, which was modeled usingI-TASSER and structural homology with bacterial dynamin-like protein(closed conformation), and OPA-1 (open conformation; see e.g., FIG. 1).As shown in the structural modeling of MFN2 in FIG. 1, MFN2 may be inits putative closed (left, inactive) and open (right, active)conformations. Critical peptide-peptide interactions betweenalpha-helices in MFN2 heptad repeat region 1 (HR1) and MFN2 heptadrepeat region 2 (HR2) are expanded in red balloon inset. HR1 367-384(inset) is agonist peptide MP-1 (Franco et al Nature 2016), whichcompetes with endogenous peptide-peptide interactions at HR2 to forceMFN1 and MFN2 opening and activation. The model shows how the firstheptad repeat domain (HR1) interacts in an anti-parallel manner with thecarboxyl terminal second heptad repeat (HR2) domain to restrain proteinunfolding and extension into the cytosol, which is a prerequisite formitochondrial fusion and trafficking (see e.g., FIG. 1). The amino acidsnecessary for the HR1-HR2 interaction were identified as Met376, His380,and Met 381 by first defining a minimal HR1-derived peptide thatcompetes with endogenous HR1-HR2 binding, followed by functionalanalyses of a complete series of alanine substituted peptides (Rocha etal 2018 Science 360:336). Based on these results, a pharmacophore modelwas developed to screen and identify chemical peptidomimetics that mimicthe 3-dimensional spatial and charge characteristics of these criticalamino acid side chains. Phosphorylation of Ser378 on the HR1 domainregulates the orientation of Met376, His380, and Met 381, regulating thepeptide-peptide interaction that maintains the closed proteinconformation.

Example 2: Chemical Peptidomimetics of the Minimal MFN2 HR1 Peptide Maybe Mitofusin Activators

Screening of commercially available compounds that conformed to themini-peptide pharmacophore model identified compounds A and B. compoundA (EC50 about 30 nM) was markedly less effective and potent as amitofusin activator than compound B (EC50 about 10 nM) (Rocha et al 2018Science 360:336).

The efficacy and synergy of the prototype mitofusin activators wereenhanced (EC50 about 3 nM) by engineering chimeric compoundsincorporating features of the parental compound A and B molecules (seee.g., TABLES 1, 2, and 3 in the reference). However, suboptimalpharmacokinetic characteristics of this series of compounds (e.g., rapiddegradation by liver microsomes and/or impermeability to blood brainbarrier/blood nerve barrier) precluded their use as clinicaltherapeutics.

Example 3: Chemical Modifications of Compound A Provide for MitofusinActivators with Enhanced Potency and Superior Pharmacokinetic Properties

Compound A was re-engineered to enhance its pharmacophorecharacteristics, resulting in a novel class of agents having potentfusogenic properties (EC50 about 3 nM), with an improved combination ofstability in the liver microsome assay and passive permeability, whichcorrelates with blood brain barrier permeability (see e.g., FIGS. 2-5).FIG. 2 shows improved novel mitofusin agonists modified from parentcompound A (Rocha Science 2018) with urea containing backbones, theright panel shows results of functional screening; DMSO is vehicle andReg C is positive control mitofusin agonist. MiM5 and MiM8-MiM12 arehighly active. MiM10, MiM11, and MiM12 exhibited average EC50 values of5 nm, 40.84 nM, and 5 nm, respectively. Urea backbone compounds weregenerally synthesized according to Scheme 1 below. FIG. 3 shows improvedmitofusin activators modified from parent compound A (Rocha Science2018) having amide backbones, the right panel shows results offunctional screening; DMSO is vehicle and Reg C is positive controlmitofusin agonist. MiM81, MiM101 and MiM111 exhibited average EC50values of 19.41, 46.93 and 8.18 nM, respectively. MiM121 exhibited anaverage EC50 of 6.3 nm. Amide backbone compounds were generallysynthesized by Scheme 2 below. FIG. 4 shows improved amide mitofusinactivators modified from parent compound A (Rocha Science 2018) havingcyclic backbone structures, the right panel shows results of functionalscreening; DMSO is vehicle and Reg C is positive control mitofusinagonist. All compounds except MiM111 cb2 were highly active. Cyclicbackbone compounds containing an amide were synthesized by amodification of Scheme 2 below, after obtaining an appropriatephenyl-substituted cycloalkyl carboxylic acid. MiM111cpr1, MiM111cpr2,and MiM111cb1 exhibited average EC50 values of 5.1, 5.9 and 18 nM,respectively. FIG. 5 shows improved amide mitofusin activators modifiedfrom parent compound A (Rocha Science 2018) having pyridine orpyridimidine substitutions, the right panel shows results of functionalscreening; DMSO is vehicle and Reg C is positive control mitofusinagonist. All compounds except MiM111 N1 were highly active. MiM111 N2,MiM111 N3, MiM111 N4, and MiM111 N1,N3 exhibited average EC50 values of23.15, 4.14, 1.27 and 4.58 nM, respectively. Pyridine backbone compoundscontaining an amide were likewise synthesized by a modification ofScheme 2 below, optionally after synthesizing an appropriatepyridinehexanoic acid or pyrimidinehexanoic acid (Scheme 3). Inaddition, Table 1 below shows MiM111 reversal of neuromusculardegeneration in a mouse model of neurodegenerative Charcot-Marie-Toothdisease type 2A as measured by Rotarod testing.

TABLE 1 Benchmark MiM111 Calculated Properties Molecular Weight (g/mol)<400 289.4 Calculated log P  <3  3.22 TP SA (A) <100  49.33 LE  >0.4 0.52 LLE  >5  4.82 Rotatable Bond #  <10  7 HBD  <5  4 HBD + HBA  <10 2 Fsp3  >45%   61% Functional Properties EC50 Mito Elongation (nM)  >30 9 Selectivity  >10 100-fold ∞M; 30-fold In Vivo PK/PD CNS Eliminationt_(1/2) (hours)  >3  3.4 (ss) Plasma t_(1/2) (hours)  >1  2.22 (oral)Oral Bioactivity  >50%  75.50% In Vitro DPMK Plasma Stability (120 min.)H  >90%   87% M  >90%   100% Plasma Protein % Bound H  <90%   91% M <90% 96.30% Solubility  >40 ∞M 175 ∞M Liver Microsomes t_(1/2)H >100 >145 (minutes) M >100 92.4 PAMPA (Pe, nm/s)  >10 26.277 P-gpeflux Ratio  >3  1.74 In Vitro Toxicology Cyp450 IC50 ∞M  >30 >50, all 5hERG VC (% inhib. 10 ∞M)  <25  1.56% Ames Test negative negative In VivoToxicology MTD (48 hours, mg/kg) >100 MTD (4 weeks) no liver, renal, no30 CNS mg/kg/day MTD (2 months) no liver, renal, CNS MTD (6 months) noliver, renal, CNS

This class of mitofusin activators has characteristics making itsuperior as an in vivo treatment for CMT2A, ALS, other neurodegenerativeconditions, and nerve injury.

Example 4: The Mitofusin Activator MiM111 Corrects Mitochondrial Defectsand Neuromuscular Dysfunction in Experimental Models of CMT2A

MiM111 is a pharmaceutically acceptable cyclohexanol/amide derivative ofCompound A (see e.g., FIGS. 2-5 and Table 1). In particular, Table 1above shows representative biological and physiochemical assays ofMiM111 activity on mitofusin 2 and on CMT2A neurons. Like structurallydissimilar Chimera C, MiM111 is potent (EC50 about 9 nM), induces anopen conformation of its target MFN2, and promotes neuronalregrowth/regeneration in vitro (see e.g., FIGS. 6A-6C). FIGS. 6A-6Cillustrate how MiM111 is a potent mitofusin activator and promotesneuronal regeneration. FIG. 6A shows the dose-response relations ofMiM5, MiM11, and MiM111 compared to prototype Chimera compound describedin Rocha et al Science 2018. FIG. 6B shows the MiM111 conformationalopening of mitofusin 2 mimics that of agonist peptide described inFranco et al Nature 2016. FIG. 6C shows how MiM111 promotes regrowth ofmouse Charcot-Marie-Tooth 2A dorsal root ganglion neurons in culture. Ina humanized mouse model of CMT2A wherein human mutant MFN2 T105M isexpressed in motor neurons, MiM111 acutely corrected characteristicsciatic nerve mitochondrial immobility and, when administeredchronically, reversed neuromuscular dysfunction (see e.g., FIGS. 7A-7B).FIGS. 7A-7B show an example of MiM111 reversing defects in experimentalCMT2A. In FIG. 7A, the schematics at top show experimental design; dotblots at bottom show results. The CMT2A MFN2 T105M mouse recapitulateshuman CMT2A with progressive neuromuscular dysfunction, shown here byRotarod latency (time to falling off). After murine CMT2A was fullydeveloped at 50 weeks of age, mice were randomized to treatment withMiM111 or vehicle. FIG. 7B shows the results of MiM111 having reversedRotarod defect in all treated mice within 8 weeks of treatment (thestatistics used 2-way ANOVA).

Syntheses of Phenylbutyl Urea Backbone Compounds

Phenylbutyl urea compounds were generally synthesized by Scheme 1 below.Examples 5-8 are illustrative of the syntheses of these types ofbackbone compounds.

Example 5: 1-(2-Methylcyclohexyl)-3-(4-phenylbutyl)urea (MiM11)

To a solution of 2-methylcyclohexan-1-amine (0.759 g, 6.70 mmol) andDIPEA (0.866 g, 6.70 mmol) in THF (10 mL) was added CDI (1.09 g, 6.70mmol), and the mixture was stirred at 25° C. for 10 min. To the mixturewas added 4-phenylbutan-1-amine (1.00 g, 6.70 mmol), and the mixture wasstirred at 25° C. for 16 h. The crude product was purified byreverse-phase HPLC, the eluent was concentrated under reduced pressure,the mixture was filtered, and then the filter cake was washed with asaturated solution of NaHCO₃(20 mL×2) and washed with H₂O (20 mL×2) togive the title compound (110.12 mg, 379.50 μmol, 5.72% yield, 99.4%purity) as a white solid. HPLC: RT=2.273 min, purity: 99.4%. LC-MS:RT=0.823 min, m/z=289.0 (M+1)⁺. ¹H NMR: 400 MHz MeOD δ 7.26-7.22 (m,2H), 7.18-7.13 (m, 3H), 3.14 (t, J=3.60 Hz, 3H), 2.63 (t, J=7.6 Hz, 2H),1.89-1.86 (m, 1H), 1.78-1.53 (m, 5H), 1.53-1.46 (m, 2H), 1.35-1.01 (m,5H), 1.07-0.91 (m, 3H). ¹³C NMR: MeOD δ 161.222, 143.825, 129.509,126.884, 56.169-48.164, 40.875, 40.093, 36.717, 35.935, 35.625, 31.149,30.106, 27.122, 26.975, 19.809.

Example 6: 1-(4-Hydroxy-2-methylcyclohexyl)-3-(4-phenylbutyl)urea (MiM5)

To a solution of 4-amino-3-methylcyclohexan-1-ol (1.50 g, 11.6 mmol) andDIPEA (1.00 g, 7.74 mmol, 1.35 mL) in THF (10 mL) was added CDI (1.26 g,7.74 mmol), and the reaction mixture was stirred at 25° C. for 10 min.Then, 4-phenylbutan-1-amine (1.16 g, 7.74 mmol, 1.22 mL) was added tothe reaction mixture and stirred at 25° C. for 16 h. The reactionmixture was poured into H₂O (10 mL) and extracted with EtOAc (5 mL×3),and the organic layers were dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to give a residue, which waspurified by reverse-phase HPLC to obtain the title compound (1.63 g,5.16 mmol, 66.6% yield) as a light yellow solid. HPLC: RT=2.22 min,purity: 96.3%. LC-MS: RT=1.988 min, mlz=305.2 (M+H)⁺. ¹H NMR: 400 MHzCDCl₃ δ 7.20-7.06 (m, 5H), 5.34 (s, 1H), 3.94-3.73 (m, 1H), 3.65-3.50(m, 1H), 3.10 (t, J=7.2 Hz, 2H), 2.55 (t, J=7.2 Hz, 2H), 1.74-1.09 (m,10H), 0.83 (d, J=6.8 Hz, 3H). ¹³C NMR: CDCl₃ δ 142.13, 142.06, 128.37,125.80, 77.45-76.61, 69.85, 48.46, 40.46, 38.44, 35.53, 33.79, 29.75,28.67, 18.41, 18.37.

Example 7: (4-Hydroxycyclohexyl)-3-(4-phenylbutyl)urea (MiM11)

To a solution of 4-phenylbutan-1-amine (1.0 g, 6.70 mmol 1.06 mL) andDIPEA (866 mg, 6.70 mmol, 1.17 mL) in THF (10 mL) was added CDI (1.09 g,6.70 mmol, 1.0 equiv), and the mixture stirred at 25° C. for 15 min. Tothe reaction mixture was added 4-aminocyclohexan-1-ol (772 mg, 6.70mmol) and stirred at 25° C. for 23.5 h. The reaction mixture wasconcentrated and purified by reverse-phase HPLC (column: Phenomenex lunaC18 250×50 mm x 10 μm; mobile phase: [water (0.05% HCl)-ACN]; B %:25-55%, 25 min) twice. The mixture was concentrated under reducedpressure and filtered, and the filter cake was washed with NaHCO₃ togive the title compound (620 mg, 1.62 mmol, 69.0% yield) as white solid.HPLC: RT=1.73 min, purity: 99.8%. LC-MS: RT=0.842 min, m/z=291.1 (M+1)⁺.¹H NMR: 400 MHz MeOD δ 7.26-7.22 (m, 2H), 7.18-7.13 (m, 2H), 3.53-3.42(m, 2H), 3.12 (t, J=7.0 Hz, 2H), 2.61 (d, J=7.6 Hz, 2H), 1.91 (d, J=10.0Hz, 3H), 1.65-1.61 (m, 2H), 1.50-1.46 (m, 2H), 1.35-1.33 (m, 2H),1.25-1.20 (m, 2H). ¹³C NMR: MeOD δ 160.800, 143.788, 129.554, 129.430,126.875, 70.654, 40.842, 36.688, 34.998, 32.558, 31.083, 30.069.

Syntheses of Phenylhexanamide Backbone Compounds

Phenylhexanamide compounds were generally synthesized by Scheme 2 below.Examples 8-11 are illustrative of the syntheses of these types ofbackbone compounds.

Example 8: N-(Tetrahydro-2H-pyran-4-yl)-6-phenylhexanamide (MiM081)

Hydroxybenzotriazole (HOBt, 253 mg, 1.87 mmol, 1.20 eq.),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCl, 448 mg, 2.34 mmol,1.50 eq.), and N,N-diisopropylethylamine (DIEA, 403 mg, 3.12 mmol, 543μL, 2.00 eq.) were added to a solution of 6-phenylhexanoic acid (0.30 g,1.56 mmol, 294 μL, 1.00 eq.) in DMF (3.0 mL). Then,tetrahydro-2H-pyran-4-amine (173 mg, 1.72 mmol, 1.10 eq.) was added tothe reaction mixture. The reaction mixture was stirred at 25° C. for 3h. The reaction mixture was concentrated under reduced pressure to givea residue. The residue was purified by preparative HPLC. The titlecompound (169.36 mg; 38% yield) was obtained as a white solid. MS:m/z=276.0 (M+H)⁺. ¹H NMR (400 MHz): DMSO-d₆ δ 7.72 (d, J=7.58 Hz, 1H)7.08-7.33 (m, 5H) 3.65-3.86 (m, 3H) 3.31-3.36 (m, 2H) 2.55 (t, J=δ 0.64Hz, 2H) 2.03 (t, J=δ 0.40 Hz, 2H) 1.64 (m, 2H) 1.53 (m, 4H) 1.20-1.40(m, 4H). ¹³C NMR: DMSO δ 171.713, 142.682, 128.725-128.651, 126.043,66.383, 45.129, 35.811-35.526, 33.039, 31.213, 28.661, 25.629.

Example 9: N-(Piperidin-4-yl)-6-phenylhexanamide (MiM091)

HOBt (422 mg, 3.12 mmol, 1.20 eq.), EDCl (748 mg, 3.90 mmol, 1.50 eq.),and DIEA (672 mg, 5.20 mmol, 906 uL, 2.00 eq.) were added at 25° C. to asolution of tert-butyl 4-aminopiperidine-1-carboxylate (500 mg, 2.60mmol, 490 μL, 1.00 eq.) and 6-phenylhexanoic acid (573 mg, 2.86 mmol,1.10 eq.) in DMF (3.00 mL). The mixture was stirred at 25° C. for 12 h.The reaction mixture was diluted with H₂O (10.0 mL) and extracted withEtOAc (10.0 mL×2). The organic phase was adjusted to a pH of 4 with 1 MHCl and extracted with EtOAc (20.0 mL). The combined organic layers wereadjusted to a pH of 8 with aqueous NaHCO₃. The combined organic layerswere washed with brine (20.0 mL), dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to give a residue. The BOC-protectedamide (740 mg; 76% yield) was obtained as a yellow solid. MS: m/z=319.3(M+H)⁺. ¹H NMR (400 MHz): DMSO-d₆ δ 7.71 (d, J=7.8 Hz, 1H), 7.30-7.21(m, 2H), 7.20-7.11 (m, 3H), 3.81 (br d, J=12.4 Hz, 2H), 3.73-3.62 (m,1H), 2.90-2.71 (m, 2H), 2.54 (t, J=7.6 Hz, 3H), 2.02 (t, J=7.4 Hz, 2H),1.69-1.60 (m, 2H), 1.59-1.46 (m, 4H), 1.39 (s, 9H), 1.28-1.11 (m, 3H).

HCl/MeOH (4 M, 12 mL, 24.3 eq.) was added to a solution of theBOC-protected amide (740 mg, 1.98 mmol, 1.00 eq. in MeOH (3 mL). Themixture was stirred at 25° C. for 16 h. The mixture was concentratedunder reduced pressure to give a residue. The residue was purified bypreparative HPLC. The title compound (418.27 mg; 73% yield) was obtainedas a white solid. MS: m/z=275.1 (M+H)⁺. ¹H NMR (400 MHz): MeOD δ7.25-7.22 (m, 2H), 7.17-7.13 (m, 3H), 3.81-3.76 (m, 1H), 3.14-3.11 (m,2H), 2.79-2.78 (m, 2H), 2.76-2.75 (m, 2H), 2.60 (t, J=7.2 Hz, 2H), 2.16(t, J=7.2 Hz, 2H), 1.88-1.85 (m, 2H), 1.65-1.61 (m, 2H), 1.61-1.46 (m,4H), 1.46-1.35 (m, 2H), 1.35-1.33 (m, 2H). ¹³C NMR: MeOD δ 175.676,143.873, 129.713, 129.582, 129.419, 126.827, 47.291, 45.424, 37.125,36.856, 32.519, 32.274, 29.829, 27.040.

Example 10: N-(4,4-Difluorocyclohexyl)-6-phenylhexanamide (MiM101)

HOBt (253 mg, 1.87 mmol, 1.20 eq.), EDCl (448 mg, 2.34 mmol, 1.50 eq.),and DIEA (403 mg, 3.12 mmol, 543 μL, 2.00 eq.) were added to a solutionof 4,4-difluorocyclohexylamine (300 mg, 1.56 mmol, 294 μL, 1.00 eq.) inDMF (3.0 mL). Then, 6-phenylhexanoic acid (232 mg, 1.72 mmol, 1.10 eq.)was added to the mixture. The mixture was stirred at 25° C. for 3 h. Themixture was concentrated under reduced pressure to give a residue. Theresidue was purified by preparative HPLC. The title compound (98.23 mg;20% yield) was obtained as a white solid. MS: m/z=310.1 (M+H)⁺. ¹H NMR(400 MHz): DMSO-d₆ δ 7.72 (d, J=7.58 Hz, 1H), 7.13-7.28 (m, 5H), 3.72(d, J=7.58 Hz, 1H), 2.55 (t, J=7.58 Hz, 2H), 2.01-2.06 (m, 2H),1.68-2.00 (m, 6H), 1.48-1.61 (m, 4H), 1.39-1.48 (m, 2H), 1.24 (m, 2H).¹³C NMR: DMSO-d₆ δ 171.949, 142.682, 128.733-128.659, 126.059, 45.072,35.778, 35.518, 31.727, 31.197, 28.621, 28.482, 28.384, 25.620.

Example 11: N-(4-hydroxycyclohexyl)-6-phenylhexanamide (MiM111)

The chemical synthesis of an advanced lead, designated MiM111, isdescribed here and illustrated in Scheme 2-4 and Scheme 2-5 below.

To a solution of 4-(t-butoxycarbonylamino)cyclohexanol (9.00 g, 41.8mmol, 1.00 eq.) in ethyl acetate (18.0 mL) was added HCl/dioxane (4 M,36.0 mL, 3.44 eq.). The mixture was stirred at 20° C. for 1 h. Thinlayer chromatography (TLC, petroleum ether/ethyl acetate=1/2) showedcomplete consumption of the starting material (R_(f)=0.50), and a newmain spot (R_(f)=0.02) was formed. The mixture was filtered and theresulting filter cake was washed with ethyl acetate (10.0 mL×3),filtered, and concentrated under reduced pressure to obtain a residue,which was used in the next step without further purification.4-aminocyclohexanol hydrochloride (6.08 g, 40.1 mmol, 95.9% yield, HClsalt) was obtained as off-white solid.

Phenylhexanoic acid (6.92 g, 36.0 mmol, 6.78 mL, 1.00 eq.), HOBt (5.83g, 43.2 mmol, 1.20 eq.) and EDCl (10.3 g, 54.0 mmol, 1.50 eq.) wereadded to a solution of 4-aminocyclohexanol hydrochloride (6.00 g, 39.6mmol, 1.10 eq., HCl salt) and DIEA (14.0 g, 108 mmol, 18.8 mL, 3.00 eq.)in N,N-dimethylformamide (DMF, 60.0 mL). The mixture was stirred at 25°C. for 16 h. Liquid chromatography with mass spectrometric detection(LCMS, EW18054-2-P1A3) showed complete consumption of the phenylhexanoicacid and the desired MS(R_(t)=0.684 min, 0.709 min) was detected. Thereaction mixture was diluted with ethyl acetate (300 mL) and washed withsaturated brine (150 mL×5). The combined organic layer was washed with1N HCl (48.0 mL), dried over anhydrous Na₂SO₄, filtered, and thefiltrate was concentrated under reduced pressure to obtain a residue.The residue was purified by preparative HPLC (column: Phenomenex LUNA®C18 250×80 mm×10 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B %: 38%ACN—68% ACN, 8.5 min) to obtain a residue following solvent removal. Theresidue was triturated with a 5:1 mixture of petroleum ether:ethylacetate (240 mL) at 25° C. for 5 minutes to obtain MiM111 (3.53 g, 12.0mmol, 33.9% yield, 99.93% purity) as an off-white solid.

After drying, products were characterized by LCMS, ¹H NMR, and ¹³C NMR.HPLC: EW18054-2-P1A, product: R_(t)=10.792 min, purity: 99.93% under 210nm.

High-resolution mass spectrometry (HRMS): RT=1.884 min, m/z=290.2128(M+H)⁺. ¹H NMR: 400 MHz MeOD δ 7.25-7.22 (m, 2H), 7.16-7.11 (m, 3H),3.59-3.48 (m, 2H), 2.60 (t, J=7.6 Hz, 2H), 2.13 (t, J=7.4 Hz, 2H),1.92-1.65 (m, 4H), 1.65-1.59 (m, 4H), 1.35-1.29 (m, 6H)¹³C NMR: MeOD δ175.669, 143.887, 129.595, 126.817, 70.605, 37.240, 36.869, 34.973,32.517, 31.668, 29.814, 27.102.

Syntheses of Pyridylhexanamide or Pyrimidinylhexanamide BackboneCompounds

Pyridylhexanamide or pyrimidinylhexanamide compounds were generallysynthesized by a modification of Scheme 2 above. Pyrimidinylhexanoicacid was generally synthesized by Scheme 3 below. Examples 12-15 areillustrative of the syntheses of these types of backbone compounds.

Example 12: (4-Hydroxycyclohexyl)-6-(pyridin-2-yl)hexanamide (MiM111 N1)

To a solution of 6-(pyridin-2-yl)hexanoic acid (1.09 g, 5.64 mmol) inDMF (10 mL) were added HOBt (914 mg, 6.77 mmol), EDCl (1.62 g, 8.46mmol), and DIEA (2.19 g, 16.9 mmol, 2.95 mL). Then,4-aminocyclohexan-1-ol (940 mg, 6.20 mmol, 1.10 equiv, HCl) was added tothe mixture and stirred at 25° C. for 16 h, and the residue was purifiedby reverse-phase HPLC (column: Waters Xbridge C18 150×50 mm×10 μm;mobile phase: [water (10 mM NH4HCO3)-ACN]; B %: 7-37%, 10 min) to obtainthe title compound (106.1 mg, 355 μmol, 6.30% yield) as an off-whitesolid. HPLC: RT=1.78 min, purity: 97.2%. LC-MS: RT=0.752 min, m/z=291.0(M+H)⁺. ¹H NMR: 400 MHz MeOD, δ 8.42 (d, J=4.00 Hz, 1H), 7.77-7.73 (m,1H), 7.31-7.30 (m, 1H), 7.25-7.23 (m, 1H), 3.61-3.48 (m, 2H), 2.80-2.76(m, 2H), 2.16-2.12 (m, 2H), 1.91-1.91 (m, 2H), 1.72-1.70 (m, 2H),1.65-1.63 (m, 2H), 1.61-1.61 (m, 2H), 1.37-1.33 (m, 4H), 1.32-1.26 (m,2H). ¹³C NMR: MeOD. δ 175.538, 163.333, 149.597, 138.868, 124.829,122.881, 70.591, 38.666, 37.133, 34.973, 31.671, 30.954, 29.861, 27.016.

Example 13: (4-Hydroxycyclohexyl)-6-(pyridin-3-yl)hexanamide (MiM111 N2)

To a solution of 6-(pyridin-3-yl)hexanoic acid (594 mg, 3.07 mmol) inDMF (10 mL) were added HOBt (498 mg, 3.69 mmol), EDCl (883 mg, 4.61mmol), and DIEA (1.19 g, 9.22 mmol, 1.61 mL). Then,4-aminocyclohexan-1-ol (512 mg, 3.38 mmol, HCl) was added to themixture, and the mixture was stirred at 25° C. for 16 h. LC-MS showedconsumption of the starting material and formation of the desiredproduct. The reaction was diluted with H₂O (20 mL) and extracted withethyl acetate (10 mL×3). The combined organic layers were washed withbrine (10 mL×3), dried over Na₂SO₄, filtered, and concentrated underreduced pressure to give a residue. The residue was purified byreverse-phase HPLC (column: Waters Xbridge C18 150×50 mm×10 μm; mobilephase: [water (10 mM NH₄HCO₃)-ACN]; B %: 7-37%, 10 min) to afford thetitle compound (100.5 mg, 346 μmol, 11.2% yield) as an off-white solid.HPLC: RT=1.53 min, purity: 99.9%. LC-MS: product: RT=0.763 min,m/z=291.0 (M+H)⁺. ¹H NMR: 400 MHz MeOD, δ 8.38-8.37 (m, 1H), 8.35-8.34(m, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.36-7.33 (m, 1H), 3.59-3.49 (m, 2H),2.68-2.65 (m, 2H), 2.16-2.12 (m, 2H), 1.92-1.92 (m, 2H), 1.68-1.65 (m,2H), 1.65-1.61 (m, 4H), 1.36-1.35 (m, 4H), 1.32-1.26 (m, 2H). ¹³C NMR:MeOD δ 175.513, 150.200, 147.599, 140.262, 138.436, 125.286, 70.575,37.109, 34.973, 33.734, 32.038, 31.671, 29.690, 26.926.

Example 14: (4-Hydroxycyclohexyl)-6-(pyridin-4-yl)hexanamide (MiM111 N3)

To a solution of 6-(pyridin-4-yl)hexanoic acid (100 mg, 517 μmol) in DMF(1.00 mL) were added EDCl (149 mg, 776 μmol), HOBt (83.9 mg, 621 μmol),and DIEA (201 mg, 1.55 mmol, 270 μL). Then, 4-aminocyclohexan-1-ol (86.3mg, 569 HCl) was added to the mixture and stirred at 25° C. for 16 h.LC-MS showed consumption of the starting material and formation of thedesired product. The mixture was purified by reverse-phase HPLC (column:Waters Xbridge C18 150×25 mm×5 μm; mobile phase: [water (10 mMNH₄HCO₃)-ACM; B %: 8-38%, 10 min), and the title compound (43.73 mg, 149μmol, 28.9% yield) was obtained as a white solid. HPLC: RT=1.50 min,purity: 99.7%. LC-MS: RT=0.748 min, m/z=291.1 (M+H)⁺. ¹H NMR: 400 MHzMeOD, δ 8.40-8.38 (m, 2H), 7.29-7.27 (m, 2H), 3.61-3.48 (m, 2H), 2.67(t, J=7.6 Hz, 2H), 2.14 (t, J=7.2 Hz, 2H), 1.95-1.91 (m, 2H), 1.87-1.84(m, 2H), 1.68-1.61 (m, 4H), 1.36-1.35 (m, 2H), 1.35-1.26 (m, 4H). ¹³CNMR: MeOD, δ 175.505, 154.685, 149.937, 125.853, 125. 853, 70.580,37.100, 36.045, 34.965, 31.668, 31.182, 29.723, 26.896.

Example 15: (4-Hydroxycyclohexyl)-6-(pyrimidin-4-yl)hexanamide (MiM111N1,N3)

To a solution of 4-chloropyrimidine (1.00 g, 6.62 mmol, HCl), CuI (63.1mg, 331 μmol), Pd(PPh3)₂Cl₂ (232 mg, 331 μmol), and TEA (2.01 g, 19.9mmol, 2.77 mL) in DMF (10.0 mL) was added methyl hex-5-ynoate (835 mg,6.62 mmol, 1.00 equiv). The mixture was stirred at 20° C. for 16 h, andmethyl 6-(pyrimidin-4-yl)hex-5-ynoate (740 mg, 3.33 mmol, 50.3% yield,91.9% purity) was obtained as a yellow oil.

To a solution of 6-(pyrimidin-4-yl)hex-5-ynoate (740 mg, 3.33 mmol, 1.00equiv) in MeOH (10.0 mL) was added Pd/C (74.0 mg, 10%) under N2. Themixture was stirred at 25° C. for 16 h under H2 (15 psi), and methyl6-(pyrimidin-4-yl)hexanoate (734 mg, crude) was obtained as a yellowoil.

To a solution of methyl 6-(pyrimidin-4-yl)hexanoate (734 mg, 3.52 mmol,1.00 equiv) in THF (3.00 mL), MeOH (3.00 mL), and H₂O (3.00 mL) wasadded LiOH.H₂O (296 mg, 7.05 mmol, 2.00 equiv). The mixture was stirredat 25° C. for 16 h, and 6-(pyrimidin-4-yl)hexanoic acid (1.20 g, crude)was obtained as a yellow solid.

To a solution of 6-(pyrimidin-4-yl)hexanoic acid (1.20 g, 6.18 mmol) inDMF (12.0 mL) were added EDCl (1.78 g, 9.27 mmol), HOBt (1.00 g, 7.41mmol), and DIPEA (2.40 g, 18.5 mmol, 3.23 mL). Then,4-aminocyclohexan-1-ol (1.03 g, 6.80 mmol, HCl) was added to themixture, and the mixture was stirred at 25° C. for 16 h. LC-MS showedconsumption of the starting material and formation of the desiredproduct. The mixture was diluted with H₂O (50 mL) and extracted withEtOAc (50 mL×3). The combined organic layers were dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give a residue. Theresidue was purified by reverse-phase HPLC (column: Phenomenex GeminiC18 250×50 mm x 10 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B %:10-30%, 15 min), and the title compound (90.98 mg, 312 μmol, 5.05%yield) was obtained as an off-white solid. HPLC: RT=1.14 min, purity:99.9%. LC-MS: RT=0.657 min, m/z=292.1 (M+H)⁺. ¹H NMR: 400 MHz MeOD, δ9.02 (s, 1H), 8.65 (d, J=5.2 Hz, 1H), 7.43 (d, J=5.2 Hz, 1H), 3.59-3.48(m, 2H), 2.79 (t, J=7.6 Hz, 2H), 2.15 (t, J=7.6 Hz, 2H), 1.95-1.92 (m,2H), 1.88-1.84 (m, 2H), 1.80-1.72 (m, 2H), 1.68-1.60 (m, 2H), 1.40-1.35(m, 2H), 1.33-1.19 (m, 4H). ¹³C NMR: MeOD, δ 175.475, 172.851, 159.152,158.089, 122.490, 70.588, 38.344, 37.050, 34.965, 31.676, 29.781,29.682, 26.888.

Example 16: A Live Cell Mitochondrial Fusion Assay for High ThroughputScreening and Evaluation of Candidate Fusogenic Compounds

The following examples describe a live-cell fusogenicity assay suitablefor directly measuring modulated mitochondrial fusion, and theconsequences of mitofusin activation on mitochondria, in ahigh-throughput manner. This assay is superior to previous read-outs forfusogenicity of compounds, such as mitochondrial aspect ratio/elongationmeasured by confocal imaging, and mitofusin conformation assessed byFRET (Rocha et al 2018 Science 360:336), because it directly measuresfusion of mitochondria whereas the conventional assays only infer fusionfrom indirect measures.

The small molecules described herein enhance mitochondrial fusion bydestabilizing the folded conformation of MFN1 or MFN2, thus promotinginitial tethering and subsequent membrane fusion between neighboringmitochondria. Mitochondrial fusion is essential for, and activelypromotes, exchange of mitochondrial contents including protein, lipids,and DNA. The live cell assay quantifies mitochondrial fusion bymeasuring the time-dependent rate of mitochondrial protein exchange.This assay was specifically designed to screen for and evaluate thefusogenic properties (the ability of an agent to promote mitochondrialfusion) of candidate agents of any chemical class, or molecules withspecific alternate compositions, including large libraries of syntheticor natural compounds.

Mitochondrial elongation, typically reported as the increase inmitochondrial aspect ratio (long axis dimension/short axis dimension),is a standard indirect metric of mitochondrial fusion. The live cellfusion assay simultaneously measures mitochondrial elongation/aspectratio, permitting concomitant dual read-outs (e.g., mitochondrialcontent exchange and mitochondrial elongation) of mitochondrial fusion.

Mitochondrial tethering and outer membrane fusion may be evoked byactivated MFN1, MFN2, or both. The live cell fusion assay is designed todetermine if altered fusion is mediated by mitofusins, and if fusogeniccompounds affect MFN1 and MFN2 differently, by measuring mitochondrialcontent exchange and elongation stimulated by screening compounds incell with both MFN1 and MFN2, is cell having only one or the other MFN,and in cells totally lacking MFN activity.

Mitochondrial fusion in live cells has been demonstrated by assayingcontent exchange of cells with adenovirus-promoted expression of eithermitochondrial-targeted green fluorescent protein (mito-GFP) ormitochondrial-targeted red fluorescent protein (mito-RFP) afterpoly-ethylene glycol (PEG) mediated cell fusion (Franco et al Nature2016, 540:74). This PEG fusion assay system was limited by variabletransient expression of the adenoviral mitochondrial targetedfluorescent proteins, highly variable cell fusion in response to PEG,and extremely low throughput (single cell analysis over time usingconfocal imaging).

These problems were solved by engineering the photo-switchable(green/red) mitochondrial-targeted fluorophore mito-Dendra2 (Pham et alGenesis 2012, 50:833) into a lentiviral expression vector for permanent,constitutive expression. Murine fibroblasts expressing both MFN1 andMFN2, MFN1 only, MFN2 only, or neither MFN1 nor MFN2 were transformedwith the mito-Dendra2 lentivirus, propogated for 2 weeks, and selectedfor high level expression by fluorescence-activated cell sorting (FACS).Individual mito-Dendra-2 expressing cell lines with different mitofusinexpression profiles were cloned and propogated for use in the screeningassay.

The live cell mitochondrial fusion assay is initiated by patterned(e.g., 2×2 micron, 3×3 micron, 4×4 micron, etc) green-to-redmitochondrial photoswitching of cells using 405 nm frequency laserlight. FIG. 8 is a schematic depiction of a patterned photo switchingmethod for screening mitofusin-dependent mitochondrial fusion activityof small molecules, peptides, or nucleic acids. Patterned photoswitchingis achieved using either a programmable microminiaturized mirror array(e.g., Polygon400 made by MIGHTEX) (see e.g., FIG. 8) for highthroughput cell imaging or microscopy platforms, or using pixel scanningon standard confocal microscopes. Immediately after photoswitching,cells are treated with candidate fusogenic agents, vehicle (negativecontrol), or positive control mitofusin activators. Two hoursthereafter, or at different time points for time course analyses, cellsare imaged for green mitochondrial fluorescence (488 nm excitation/535nm emission) and red mitochondrial fluorescence (560 nm excitation/645nm emission) using a high-throughput imaging system or confocal orstandard fluorescence microscope. FIG. 9 shows an example of patternedphoto switching (interrupted rectangle, middle panel) screen formitochondrial fusion. Before photoswitching (left, D) cells expressingMito Dendra-2 have green fluorescence (shown as bright white).Immediately after patterned 405 nm laser illumination (middle, E), thephoto switching convers half of mitochondria to red fluorescence (shownas darker grey). Over time (15 minutes; right panel, F), green and redmitochondria will fuse, shown by arrows in merged images (right panel).The top images (A, B, and C) are enlarged from bottom (solid squares).Mitochondrial content exchange (i.e., fusion) is the overlap between redand green mitochondrial signals, which may be visualized as yellowfluorescence in merged images (shown as lighter grey) (see e.g., FIG.9).

Data are represented as number of red-green overlay pixels (newly fusedmitochondria) divided by red-green merged pixels (total mitochondria) ata given time point. Increased rate of fusion (red-green overly overtime) in MFN expressing cells, but not MFN null cells, reflects aspecific mitofusin activating effect. This system is useful in 12-, 24-,96-, or 384-well formats for high-throughput screening of mitofusinactivators.

Materials and Methods

Cell lines: Wild-type MEFs were prepared from E10.5 c57/bl6 mouseembryos. SV-40 T antigen-immortalized MFN1 null (CRL-2992), MFN2 null(CRL-2993) and MFN1/MFN2 double null MEFs (CRL-2994) were purchased fromATCC. MEFs were subcultured in DMEM (4.5 g/L glucose) plus 10% fetalbovine serum, lx nonessential amino acids, 2 mM L-glutamine, 100units/mL penicillin, and 100 μg/mL streptomycin.

Protein and Peptide Modeling: The hypothetical structures of human MFN2were developed using the I-TASSER Suite package. The putative closedconformation is based on structural homology with bacterial dynamin-likeprotein (PDB: 2J69), human MFN1 (PDB:SGNS), and Arabidopsis thalianadynamin-related protein (PDB: 3T34). The putative open conformation wasbased on structural homology with human Opal, retrieved from thefollowing structures: rat dynamin (PDB: 3ZVR), human dynamin 1-likeprotein (PDB: 4BEJ), and human myxovirus resistance protein 2 (PDB:4WHJ). Minipeptide and protein modeling used PEP-FOLD3(http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) and UCSFChimera, respectively.

Confocal Live Cell Studies of Mitochondria: Confocal imaging used aNikon Ti Confocal microscope equipped with a 60×1.3NA oil immersionobjective. All live cells were grown on cover slips loaded onto achamber (Warner instrument, RC-40LP) in modified Krebs-Henseleit buffer(138 mM NaCl, 3.7 mM KCl, 1.2 mM KH₂PO₄, 15 mM Glucose, 20 mM HEPES and1 mM CaCl₂) at room temperature.

Cells were excited with 408 nm (Hoechst), 561 nm (MitoTracker Green andCalcein AM, GFP), or 637 nm (TMRE, MitoTracker Orange, Ethidiumhomodimer-1, and AF594-Dextran) laser diodes. For mitochondrialelongation studies, mitochondrial aspect ratio (long axis/short axis)was calculated using automated edge detection and Image J software.Mitochondrial depolarization was calculated as percent of greenmitochondria visualized on MitoTracker Green and TMRE merged images,expressed as green/(green+yellow mitochondria)×100.

Analytical Methods

HPLC/HRMS (ESI): LC/MS analysis was carried out using Agilent 1100Series LC/MSD system with DAD\ELSD and Agilent LC\MSD VL (G1956A), SL(G1956B) mass-spectrometer or Agilent 1200 Series LC/MSD system withDAD\ELSD and Agilent LC\MSD SL (G6130A), SL (G6140A) mass-spectrometer.All the LC/MS data were obtained using positive/negative mode switching.The compounds were separated using a Zorbax SB-C18 1.8 μm 4.6×15 mmRapid Resolution cartridge (PN 821975-932) under a mobile phase(A—acetonitrile, 0.1% formic acid; B—water (0.1% formic acid)). Flowrate: 3 mL/min; Gradient 0 min-100% B; 0.01 min-100% B; 1.5 min-0% B;1.8 min-0% B; 1.81 min-100% B; Injection volume 1 μL; Ionization modeatmospheric pressure chemical ionization (APCI); Scan range m/z 80-1000.

Statistical Methods

Time-course and dose-response data are calculated for each study usingGraphPad Prism (La Jolla, Calif., USA). All data are reported asmean±SEM. Statistical comparisons (two-sided) used one-way ANOVA andTukey's tests for multiple groups or Student's t-test for pairedcomparisons. p<0.05 was considered significant. In vitro pharmacokineticanalyses of mitofusin activators was performed at WuXi Apptec Co. Ltd.(Shanghai, China).

Binding to human and CD-1 mouse plasma proteins was measured usingequilibrium dialysis. Pooled individual frozen EDTA anticoagulatedplasma mouse and human samples were used as test matrix. Warfarin wasused as a positive control. The test compounds were spiked into blankmatrix at the final concentration of 2 μM. A 150-μL aliquot of matrixsample was added to one side of the chamber in a 96-well equilibriumdialyzer plate (HTD dialysis) and an equal volume of dialysis buffer wasadded to the other side of the chamber. An aliquot of matrix sample washarvested before the incubation and used as T₀ samples for recoverycalculation. The incubations were performed in triplicate. The dialyzerplate was placed in a humidified incubator and rotated slowly for fourhours at 37° C. After incubation, the samples were taken from the matrixside as well as the buffer side. The plasma sample was matched withequal volume of blank buffer; and buffer samples were matched with equalvolume of blank plasma. The matrix-matched samples were quenched withstop solution containing internal standard. All samples were analyzed byLC-MS/MS. All test compound concentrations in matrix and buffer samplesare expressed as peak area ratios (PAR) of analyte/internal standard.

Activator in vitro stability was measured in human and mouse livermicrosomes. An intermediate solution (100 μM of small molecule) wasinitially prepared in methanol and subsequently used to prepare theworking solution. This was achieved by a 10-fold dilution step of theintermediate solution in 100 mM potassium phosphate buffer. Ten μL of acompound working solution or control working solution was added to allwells of a 96-well plate for the time points (minutes): T₀, T₅, T₁₀,T₂₀, T₃₀, T₆₀, NCF60, except the matrix blank. The microsome solution(680 μL/well) (#452117, Corning; Woburn, Mass., USA; #R1000, Xenotech;Kansas City, Kans., USA and #M1000, Xenotech; Kansas City, Kans., USA)was dispersed to 96-well plate as reservoir according to the plate map.Then, 80 μL/well was added to every plate by ADDA (Apricot Design DualArm, Apricot Designs, Inc., Covina, Calif., USA), and the mixture ofmicrosome solution and compound were allowed to incubate at 37° C. forabout 10 minutes. Next, 10 μL of 100 mM potassium phosphate buffer/wellwas added to NCF60 and incubated at 37° C. (timer 1H was started). Afterpre-warming, 90 μL/well of NADPH (#00616, Sigma, Aldrich, St. Louis,Mo., USA) regenerating system was dispensed to 96-well plate asreservoir according to the plate map. Then 10 μL/well was added to everyplate by ADDA to start reaction. To terminate the reaction, 300 μL/wellof stop solution (cold in 4° C., including 100 ng/mL tolbutamide and 100ng/mL labetalol as internal standards) was used, and sampling plateswere agitated for approximately 10 min. The samples were nextcentrifuged at 4000 rpm for 20 minutes at 4° C. supernatants wereanalyzed by LC-MS/MS.

Parallel artificial membrane permeability assay (PAMPA).

A 10 μM solution of a small molecule in 5% DMSO (150 μL) was added toeach well of the donor plate, whose PVDF membrane was pre-coated with 5μL of 1% brain polar lipid extract (porcine)/dodecane mixture. Then, 300μL of PBS was added to each well of the PTFE acceptor plate. The donorplate and acceptor plate were combined together and incubated for 4hours at room temperature with shaking at 300 rpm. To prepare the T₀sample, 20 μL of a donor solution was transferred to new well, followedby the addition of 250 μL PBS (DF:13.5) and 130 μL of acetonitrile (ACN)(containing internal standard) as the T₀ sample. To prepare the acceptorsample, the plate was removed from incubator and 270 μL of the solutionwas transferred from each acceptor well and mixed with 130 μL ACN(containing internal standard) as an acceptor sample. To prepare thedonor sample, 20 μL of the solution was transferred from each donor welland mixed with 250 μL PBS (DF: 13.5), 130 μL ACN (containing internalstandard) as a donor sample. The acceptor samples and donor samples wereanalyzed by LC-MS/MS.

The present invention is also directed to the following clauses.

Clause 1: A method of treating a peripheral nervous system (PNS) orcentral nervous system (CNS) genetic disorder, physical damage, and/orchemical injury, comprising:

-   -   administering to a subject a therapeutically effective amount of        a composition comprising one or more of a mitofusin activator or        a pharmaceutically acceptable salt thereof, wherein the        mitofusin activator stimulates mitochondrial fusion and enhances        mitochondrial subcellular transport.

Clause 2. The method of clause 1, wherein the composition comprises oneor more mitofusin activators, wherein the mitofusin activator comprisesa structure of formula:

-   -   or a pharmaceutically acceptable salt, tautomer, or stereoisomer        thereof,    -   wherein R¹ is selected from non-, mono-, or poly-substituted        C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, and C₃₋₈ heterocyclyl; and    -   wherein R² is selected from non-, mono-, or poly-substituted        C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, and C₃₋₈ heterocyclyl.

Clause 3. The method of clauses 1 or 2, wherein the mitofusin activatorcomprises a structure of formula:

-   -   wherein R¹ is selected from

-   -   and wherein R² is selected from

Clause 4: The method of clauses 2 or 3, wherein R¹ or R² areindependently and optionally substituted by one or more of acetamide,C₁₋₈ alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl, cyano,C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F, halo,indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, and/orthiophene;

wherein R¹ or R² are optionally further substituted with one or moreacetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl,cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ -heterocyclyl, hydroxyl,F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide,and/or thiophene; and wherein one or more of the alkyl, cycloalkyl,heteroaryl, heterocyclyl, indole, or phenyl substituent is optionallyfurther substituted with one or more of the following substituents:acetamide, alkoxy, amino, azo, Br, C₁₋₈ alkyl, carbonyl, carboxyl, Cl,cyano, C₃₋₈ cycloalkyl, C₃₋₈ heteroaryl, C₃₋₈ heterocyclyl, hydroxyl, F,halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, andthiophene.

Clause 5: The method of any one of clauses 1 to 4, wherein the mitofusinactivator is selected from:

2-(3-benzylcyclobutyl)-N-(4-hydroxycyclohexyl)acetamide.

Clause 6: The method of any of clauses 1 to 5, wherein the mitofusinactivator is a compound of Formula III

-   -   or a pharmaceutically salt thereof, wherein:    -   X is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl;    -   Z is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl;    -   R¹ and R² are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl; or R¹ and R² are taken together to form a C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R³ and R⁴ are independently selected from H, F, alkyl, COR⁷,        C₃₋₇ cycloalkyl; or R³ and R⁴ are taken together to form a C₃₋₇        cycloalkyl or heterocycloalkyl;    -   Y is selected from O, CR⁵R⁶, CR⁷═CR⁸, a triple bond, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, NR⁷, S, SO₂, SONR⁸,        —NR⁸SO₂—, —NR⁷CO—, —CONR⁷—, and —NR⁷CONR⁸—;    -   R⁵ and R⁶ are independently selected from H, F, alkyl, and        cycloalkyl; or R⁵ and R⁶ are taken together to form C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl;    -   R⁸ is selected from H, alkyl, COR⁷, and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4, or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4, or 5, wherein when o is equal to or greater        than 1, then Y═NR⁷, S, SO₂, SONR⁸, —NR⁸SO₂—, —NR⁷CO—, —CONR⁷—,        —NR⁷CONR⁸—, and wherein the sum of o+p+q is not less than 3 or        greater than 7.

Clause 7: The mitofusin activator of clause 6 or a pharmaceuticallyacceptable salt thereof, wherein:

-   -   X is selected from cycloalkyl, and heterocycloalkyl;    -   Z is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl;    -   Y is selected from O, CR⁵R⁶, cycloalkyl, and aryl;    -   R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently selected        from H and alkyl;    -   o is 0, 1, 2, 3, 4, or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4, or 5; and, wherein when o is equal to or        greater than 1, then Y is S or SO₂; and    -   wherein the sum of o+p+q is not less than 3 or greater than 7.

Clause 8: The mitofusin activator of clauses 6 or 7 or apharmaceutically acceptable salt thereof, wherein:

-   -   X is selected from a cycloalkyl with having one, two, or three        substituents independently selected from R⁷, OR⁷, NR⁷R⁸,        fluorine, and CF₃; and a heterocycloalkyl containing one or two        optionally substituted heteroatoms independently selected from        O, NR⁷, and S;    -   Z is selected from aryl and heteroaryl;    -   Y is selected from O, CH₂, and cycloalkyl;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl;    -   R⁸ is selected from H, alkyl, and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4, or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4, or 5; and, wherein when o is equal to or        greater than 1, then Y is S or SO₂; and    -   wherein the sum of o+p+q is not less than 3 or greater than 5.

Clause 9: The mitofusin activator of any of clauses 6 to 8, or apharmaceutically acceptable salt thereof, wherein

-   -   X is a cycloalkyl with one, two, or three substituents        independently selected from the group consisting of R⁷, OR⁷,        NR⁷R⁸, fluorine, and CF₃ or X is a heterocycloalkyl containing        one or two optionally substituted heteroatoms independently        selected from O, NR⁷, and S;    -   Z is selected from aryl and heteroaryl;    -   Y is selected from cyclopropyl and cyclobutyl;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl; and R⁸ is        selected from H, alkyl, COR^(i), and C₃₋₇ cycloalkyl; or R⁷ and        R⁸ are taken together to form C₃₋₇ cyclolkyl;    -   o is 0, 1, 2, or 3;    -   p is 1; and    -   q is 0, 1, 2, or 3, wherein the sum of o+p+q is not less than 3        or greater than 5.

Clause 10: The mitofusin activator of any of clauses 6 to 9 or apharmaceutically acceptable salt thereof, wherein:

-   -   X is cycloalkyl with one, two, or three substituents        independently selected from the group consisting of R⁷, OR⁷,        NR⁷R⁸, fluorine, and CF₃ or X is heterocycloalkyl containing one        or two optionally substituted heteroatoms independently selected        from O, NR⁷, and S;    -   Z is selected from aryl and heteroaryl;    -   Y is selected from O and CH₂;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ and R⁸ are independently selected from H, alkyl, and C₃₋₇        cycloalkyl; or R⁷ and R⁸ are taken together to form C₃₋₇        cyclolkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4, wherein the sum of o+p+q is 5.

Clause 11. The mitofusin activator of any of clauses 6 to 10 or apharmaceutically acceptable salt thereof, wherein

-   -   X is selected from 4-hydroxylcyclohexyl, 4-aminocyclohexyl,        4-(N-methyl)aminocyclohexyl, 4-(N,N-dimethyl)aminocyclohexyl,        4-(N-acetylamino)cyclohexyl, 4,4-difluorocyclohexyl,        tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl,        N-methyl-piperidinyl, and N-acetyl-piperidinyl;    -   Z is selected from aryl and heteroaryl;    -   Y is selected from O and CH₂;    -   R¹, R², R³, and R⁴ are each H;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4; and, wherein the sum of o+p+q is 5.

Clause 12: The mitofusin activator of any of clauses 6 to 11 or apharmaceutically acceptable salt thereof, wherein:

-   -   X is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl; each independently having has zero to four        substituents independently selected from R⁷, OR⁷, Cl, F, —CN,        CF₃, —NR⁷R⁸, —SO₂NR⁷R⁸, —NR⁷SO₂R^(g), —SO₂R^(g), —CONR⁷R⁸,        —NR⁷COR^(g), C₃₋₇ cycloalkyl, and heterocycloalkyl, wherein the        heterocycloalkyl and heteroaryl independently include one to        four heteroatoms selected from the group consisting of nitrogen,        oxygen, and sulfur;    -   Z is selected from phenyl and heteroaryl; each having zero to        four substituents independently selected from R⁷, OR⁷, Cl, F,        —CN, CF₃, —NR⁷R⁸, —SO₂NR⁷R⁸, —NR⁷SO₂R^(g), —SO₂R^(g), —CONR⁷R⁸,        —NR⁷COR^(g), C₃₋₇ cycloalkyl, and heterocycloalkyl and wherein        the heteroaryl contains one to four atoms independently selected        from nitrogen, oxygen and sulfur, and wherein the phenyl or        heterocyclic moiety;    -   Y is selected from O and CH₂;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl; and R⁸ is        selected from H, alkyl, COR⁷, and C₃₋₇ cycloalkyl; or R⁷ and R⁸        are taken together to form C₃₋₇ cyclolkyl;    -   R⁹ is selected from alkyl and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1;    -   q is 0, 1, 2, 3, or 4; and    -   wherein the sum of o+p+q is 5.

Clause 13: The mitofusin activator of any of clauses 6 to 12 or apharmaceutically acceptable salt thereof, wherein

-   -   X is selected from 4-hydroxylcyclohexyl, 4-aminocyclohexyl,        4-(N-methyl)aminocyclohexyl, 4-(N,N-dimethyl)aminocyclohexyl,        4-(N-acetylamino)cyclohexyl, 4,4-difluorocyclohexyl,        tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl,        N-methyl-piperidinyl, and N-acetyl-piperidinyl;    -   Z is selected from phenyl and heteroaryl; wherein the        heterocyclic moiety contains 1 to 3 atoms independently selected        from nitrogen, oxygen and sulfur, and wherein the phenyl or        heterocyclic moiety has O to 3 substituents independently        selected from R⁷, OR⁷, Cl, F, —CN, CF₃, —NR⁷R⁸, —SO₂R^(g),        —CONR⁷R⁸, —NR⁷COR^(g), C₃₋₇ cycloalkyl, and heterocycloalkyl;    -   Y is selected from O and CH₂;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl; and R⁸ is        selected from H, alkyl, COR⁷ and C₃₋₇ cycloalkyl; or R⁷ and R⁸        are taken together to form C₃₋₇ cyclolkyl;    -   R⁹ is selected from alkyl and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4, wherein the sum of o+p+q is 5.

Clause 14: The mitofusin activator of any of clauses 6 to 13 or apharmaceutically acceptable salt thereof, wherein

-   -   X is selected from 4-hydroxylcyclohexyl, 4-aminocyclohexyl,        4-(N-methyl)aminocyclohexyl, 4-(N,N-dimethyl)aminocyclohexyl,        4-(N-acetylamino)cyclohexyl, 4,4-difluorocyclohexyl,        tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl,        4-N-methyl-piperidinyl, and 4-N-acetyl-piperidinyl;    -   Z is selected from phenyl, 2-pyridinyl, 3-pyridinyl,        4-pyridinyl, 6-pyrimidinyl, 5-pyrimidinyl, 4-pyrimidinyl, and        2-pyrimidinyl, wherein the phenyl, pyridinyl, and pyrimidinyl        moiety has zero to two substituents independently selected from        the group consisting of R⁷, OR⁷, Cl, F, —CN, CF₃, —NR⁷R⁸,        —SO₂R^(g), —CONR⁷R⁸, and —NR⁷COR^(g),    -   Y is O or CH₂;    -   R¹, R², R³, and R⁴ are each H;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl; and R⁸ is        selected from H, alkyl, COR⁷, and C₃₋₇ cycloalkyl; or R⁷ and R⁸        are taken together to form C₃₋₇ cyclolkyl;    -   R⁹ is selected from alkyl and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4, wherein the sum of o+p+q is 5.

Clause 15: A method of treating a disease for which a mitofusinactivator is indicated, the method comprising administering to a mammalin need thereof a therapeutically effective amount of a compound ofFormula III

-   -   or a pharmaceutically salt thereof, wherein:    -   X is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl;    -   Z is selected from cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl;    -   R¹ and R² are independently selected from H, F, alkyl, and C₃₋₇        cycloalkyl; or R¹ and R² are taken together to form a C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R³ and R⁴ are independently selected from H, F, alkyl, COR⁷, and        C₃₋₇ cycloalkyl or R³ and R⁴ are taken together to form a C₃₋₇        cycloalkyl or heterocycloalkyl;    -   Y is selected from O, CR⁵R⁶, CR⁷═CR⁸, a triple bond, cycloalkyl,        heterocycloalkyl, aryl, heteroaryl, NR⁷, S, SO₂, SONR⁸,        —NR⁸SO₂—, —NR⁷CO—, —CONR⁷—, and —NR⁷CONR⁶—;    -   R⁵ and R⁶ are independently selected from H, F, alkyl, and        cycloalkyl or R⁵ and R⁶ are taken together to form C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R⁷ is selected from H, alkyl, and C₃₋₇ cycloalkyl;    -   R⁸ is selected from H, alkyl, COR⁷, and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4, or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4, or 5, wherein when o is equal to or greater        than 1, then Y═NR⁷, S, SO₂, SONR⁸, —NR⁸SO₂—, —NR⁷CO—, —CONR⁷—,        —NR⁷CONR⁸—, and wherein the sum of o+p+q is not less than 3 or        greater than 7.

Clause 16: The method of any of clauses 1 to 15, wherein the PNS or CNSdisorder is selected from any one or a combination of:

-   -   a chronic neurodegenerative condition wherein mitochondrial        fusion, fitness, or trafficking are impaired;    -   a disease or disorder associated with mitofusin 1 (MFN1) or        mitofusin 2 (MFN2) dysfunction;    -   a disease associated with mitochondrial fragmentation,        dysfunction, or dysmotility;    -   a degenerative neuromuscular condition such as        Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,        Huntington's disease, Alzheimer's disease, Parkinson's disease;    -   hereditary motor and sensory neuropathy, autism, autosomal        dominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig's        disease, cancer, mitochondrial myopathy, diabetes mellitus and        deafness (DAD), Leber's hereditary optic neuropathy (LHON),        Leigh syndrome, subacute sclerosing encephalopathy, Neuropathy,        ataxia, retinitis pigmentosa, and ptosis (NARP), myoneurogenic        gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with        ragged red fibers (MERRF), mitochondrial myopathy,        encephalomyopathy, lactic acidosis, stroke-like symptoms        (MELAS), mtDNA depletion, mitochondrial neurogastrointestinal        encephalomyopathy (MNGIE), dysautonomic mitochondrial myopathy,        mitochondrial channelopathy, or pyruvate dehydrogenase complex        deficiency (PDCD/PDH);    -   diabetic neuropathy;    -   chemotherapy-induced peripheral neuropathy; and/or    -   crush injury, spinal cord injury (SCI), traumatic brain injury        (TBI), stroke, optic nerve injury, and related conditions that        involve axonal disconnection.

Clause 17: The method of any of clauses 1 to 16, with the proviso thatthe mitofusin activator is not selected from the following compounds:

Clause 18: The method according to any of clauses 1 to 17, wherein thecomposition further comprises a pharmaceutically acceptable excipient.

Clause 19: A method of treating a CNS or PNS genetic or non-geneticneurodegenerative condition, injury, damage, or trauma comprisingadministering to the subject a therapeutically effective amount of amitofusin activator of any one of clauses 2 to 18.

Clause 20: The method of clause 19, wherein the subject is diagnosedwith or is suspected of having:

-   -   a chronic neurodegenerative condition wherein mitochondrial        fusion, fitness, or trafficking are impaired;    -   a disease or disorder associated with mitofusin 1 (MFN1) or        mitofusin 2 (MFN2) dysfunction;    -   a disease associated with mitochondrial fragmentation,        dysfunction, or dysmotility;    -   a degenerative neuromuscular condition such as        Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,        Huntington's disease, Alzheimer's disease, Parkinson's disease;    -   hereditary motor and sensory neuropathy, autism, autosomal        dominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig's        disease, cancer, mitochondrial myopathy, diabetes mellitus and        deafness (DAD), Leber's hereditary optic neuropathy (LHON),        Leigh syndrome, subacute sclerosing encephalopathy, neuropathy,        ataxia, retinitis pigmentosa, and ptosis (NARP), myoneurogenic        gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with        ragged red fibers (MERRF), mitochondrial myopathy,        encephalomyopathy, lactic acidosis, stroke-like symptoms        (MELAS), mtDNA depletion, mitochondrial neurogastrointestinal        encephalomyopathy (MNGIE), dysautonomic mitochondrial myopathy,        mitochondrial channelopathy, or pyruvate dehydrogenase complex        deficiency (PDCD/PDH);    -   diabetic neuropathy;    -   chemotherapy-induced peripheral neuropathy; and/or    -   crush injury, spinal cord injury (SCI), traumatic brain injury        (TBI), stroke, optic nerve injury, and related conditions that        involve axonal disconnection.

Clause 21: A method of screening one or more candidate molecules formitochondrial fusion modulatory activity comprising:

-   -   (i) constitutively expressing a mitochondrial-targeted        photoswitchable fluorophore in cells expressing different        combinations of MFN1 or MFN2 in a genetically defined manner;    -   (ii) photoswitching mitochondrial-targeted fluorophores in a        micro-matrix pattern in cells transiently or constitutively        expressing a mitochondrial-targeted photoswitchable fluorophore;        and    -   (iii) measuring merged/overlay fluorescence in photoswitched        mitochondria.

Clause 22: The method of clause 21, further comprising comparing themerged/overlay fluorescence of the test mixture with the merged/overlayfluorescence of the control mixture, wherein when the merged/overlayfluorescence of the test mixture is greater than the merged/overlayfluorescence of the control mixture, the one or more candidate moleculesin the test mixtures is identified as an activator of mitochondrialfusion.

Clause 23: The method of clause 21 or clause 22, further comprisingcomparing the merged/overlay fluorescence of the test mixture of acandidate agent in wild-type, MFN1, or MFN2 expressing cells with themerged/overlay fluorescence of that candidate agent in cells lackingboth MFN1 and MFN2 (MFN null cells), wherein the merged/overlayfluorescence of the mixture in MFN expressing cells is greater than themerged/overlay fluorescence of the mixture in MFN null cells, the one ormore candidate molecules in the test mixtures is identified as amitofusin activator.

The present disclosure also relates to the following embodiments:

A. Methods for treating a mitochondria-associated disease, disorder orcondition. The methods comprise: administering a therapeuticallyeffective amount of a composition comprising one or more of a mitofusinactivator or a pharmaceutically acceptable salt thereof to a subjecthaving or suspected of having a mitochondria-associated disease,disorder or condition, the mitofusin activator having a formula of

-   -   wherein:        -   X is selected from cycloalkyl and heterocycloalkyl,        -   Z is aryl;        -   R^(a) and R^(b) are independently selected from H, F, alkyl,            and C₃₋₇ cycloalkyl, or R^(a) and R^(b) taken together form            a C₃₋₇ cycloalkyl or heterocycloalkyl;        -   R^(c) and R^(d) are independently selected from H, F, alkyl,            COR^(g), and C₃₋₇ cycloalkyl, or R^(c) and R^(d) taken            together form a C₃₋₇ cycloalkyl or heterocycloalkyl;        -   Y is selected from O, CR^(e)R^(f), CR^(e)═CR^(f), C≡C            cycloalkyl, heterocycloalkyl, aryl, heteroaryl, NR^(g), S,            SO₂, SONR^(h), NR^(g)SO₂, NR^(g)CO, CONR^(g), and            NR^(g)CONR^(h);        -   R^(e) and R^(f) are independently selected from H, F, alkyl,            and cycloalkyl, or R^(e) and R^(f) taken together form C₃₋₇            cycloalkyl or heterocycloalkyl;        -   R^(g) is selected from H, alkyl, and C₃₋₇ cycloalkyl;        -   R^(h) is selected from H, alkyl, COR^(g), and C₃₋₇            cycloalkyl;        -   o is 0, 1, 2, 3, 4, or 5;        -   p is 0 or 1; and        -   q is 0, 1, 2, 3, 4, or 5, provided that if Y is cycloalkyl            and p is 1, the sum of o+p+q is not less than 3 or greater            than 5 and otherwise the sum of o+p+q is 5.

B. Compositions comprising a mitofusin activator or a pharmaceuticallyacceptable salt thereof. The compositions comprise: one or more of amitofusin activator or a pharmaceutically acceptable salt thereof, themitofusin activator having a formula of

wherein:

-   -   X is selected from cycloalkyl and heterocycloalkyl,    -   Z is aryl;    -   R^(a) and R^(b) are independently selected from H, F, alkyl, and        C₃₋₇ cycloalkyl, or R^(a) and R^(b) taken together form a C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R^(b) and R^(d) are independently selected from H, F, alkyl,        COR^(g), and C₃₋₇ cycloalkyl, or R^(b) and R^(d) taken together        form a C₃₋₇ cycloalkyl or heterocycloalkyl;    -   Y is selected from O, CR^(e)R^(f), CR^(e)═CR^(f), C≡C        cycloalkyl, heterocycloalkyl, aryl, heteroaryl, NR^(g), S, SO₂,        SONR^(h), NR^(g)SO₂, NR^(g)CO, CONR^(g), and NR^(g)CONR^(h);    -   R^(e) and R^(f) are independently selected from H, F, alkyl, and        cycloalkyl, or R^(e) and R^(f) taken together form C₃₋₇        cycloalkyl or heterocycloalkyl;    -   R^(g) is selected from H, alkyl, and C₃₋₇ cycloalkyl; R^(h) is        selected from H, alkyl, COR^(g), and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, 4, or 5;    -   p is 0 or 1; and    -   q is 0, 1, 2, 3, 4, or 5, provided that if Y is cycloalkyl and p        is 1, the sum of o+p+q is not less than 3 or greater than 5 and        otherwise the sum of o+p+q is 5.

Embodiments A and B may include one or more of the following elements inany combination:

Element 1: wherein:

-   -   Y is selected from O, CR^(e)R^(f), cycloalkyl, and aryl;    -   R^(a), R^(b), R^(c), R^(d), R^(e), and R^(f) are each        independently selected from H and alkyl; and    -   p is 1.

Element 2: wherein:

-   -   X is selected from a cycloalkyl having one, two, or three        substituents independently selected from the group consisting of        R^(g), OM, NR^(g)R^(h), F, and CF₃, and a heterocycloalkyl        containing one or two optionally substituted heteroatoms        independently selected from O, NR^(g), and S;    -   Y is selected from O, CH₂, and cycloalkyl; R^(a), R^(b), R^(c),        and R^(d) are each H; R^(g) is selected from H, alkyl, and C₃₋₇        cycloalkyl; R^(h) is selected from H, alkyl, and C₃₋₇        cycloalkyl; and p is 1.

Element 3: wherein:

-   -   X is selected from a cycloalkyl with one, two, or three        substituents independently selected from the group consisting of        R^(g), OM, NR^(g)R^(h), F, and CF₃, and a heterocycloalkyl        containing one or two optionally substituted heteroatoms        independently selected from O, NR^(g), and S;    -   Y is selected from cyclopropyl and cyclobutyl;    -   R^(g), R^(h), R^(c), and R^(d) are each H;    -   R^(g) is selected from H, alkyl, and C₃₋₇ cycloalkyl;    -   R^(h) is selected from H, alkyl, COR^(g), and C₃₋₇ cycloalkyl,        or R^(g) and R^(h) taken together form a C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, or 3;    -   p is 1; and    -   q is 0, 1, 2, or 3.

Element 4: wherein:

-   -   X is a cycloalkyl having one, two, or three substituents        independently selected from the group consisting of R^(g),        OR^(g), NR^(g)R^(h), F, and CF₃, or X is a heterocycloalkyl        containing one or two optionally substituted heteroatoms        independently selected from O, NR^(g), and S;    -   Y is selected from O and CH₂;    -   R^(a), R^(b), R^(c), and R^(d) are each H;    -   R^(g) and R^(h) are independently selected from H, alkyl, and        C₃₋₇ cycloalkyl, or R^(g) and R^(h) taken together form C₃₋₇        cycloalkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4.

Element 5: wherein X is selected from 4-hydroxycyclohexyl,4-aminocyclohexyl, 4-(N-methyl)aminocyclohexyl,4-(N,N-dimethyl)aminocyclohexyl, 4-(N-acetylamino)cyclohexyl,4,4-difluorocyclohexyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperidinyl, N-methyl-piperidinyl, and N-acetyl-piperidinyl.

Element 6: wherein x has zero to four substituents independentlyselected from R^(g), OM, Cl, F, CN, CF₃, NR^(g)R^(h), SO₂NR^(g)R^(h),NR^(g)SO₂R^(i), SO₂R^(i), CONR^(g)R^(h), NR^(g)COR^(i), C₃₋₇ cycloalkyl,and heterocycloalkyl, wherein the heterocycloalkyl includes one to fourheteroatoms selected from the group consisting of nitrogen, oxygen, andsulfur;

-   -   Z is selected from phenyl having zero to four substituents        independently selected from R^(g), OM, Cl, F, CN, CF₃,        NR^(g)R^(h), SO₂NR^(g)R^(h), NR^(g)SO₂R^(i), SO₂R^(i),        CONR^(g)R^(h), NR^(g)COR^(i), C₃₋₇ cycloalkyl, and        heterocycloalkyl;    -   Y is selected from O and CH₂;    -   R^(a), R^(b), and R^(d) are each H;    -   R^(g) is selected from H, alkyl, and C₃₋₇ cycloalkyl, and R^(h)        is selected from H, alkyl, COR^(g), and C₃₋₇ cycloalkyl, or        R^(g) and R^(h) taken together form a C₃₋₇ cycloalkyl;    -   R^(i) is selected from alkyl and C₃₋₇ cycloalkyl;    -   o is 0, 1, 2, 3, or 4;    -   p is 1; and    -   q is 0, 1, 2, 3, or 4.

Element 7: wherein the mitofusin activator has a structure of

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof,

-   -   wherein R¹ is selected from unsubstituted, mono-substituted, or        poly-substituted C₃₋₈ cycloalkyl and C₃₋₈ heterocycloalkyl; and    -   wherein R² is aryl.

Element 8: wherein R¹ is selected from:

-   -   and wherein R² is selected from:

Element 9: wherein the mitofusin activator is selected from

Element 10: wherein the mitochondria-associated disease, disorder orcondition is a peripheral nervous system (PNS) or central nervous system(CNS) genetic or non-generic disorder, physical damage, and/or chemicalinjury.

Element 11: wherein the PNS or CNS disorder is selected from any one ora combination of:

a chronic neurodegenerative condition wherein mitochondrial fusion,fitness, or trafficking are impaired;a disease or disorder associated with mitofusin 1 (MFN1) or mitofusin 2(MFN2) dysfunction;

-   -   a disease associated with mitochondrial fragmentation,        dysfunction, or dysmotility;    -   a degenerative neuromuscular condition such as        Charcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis,        Huntington's disease, Alzheimer's disease, Parkinson's disease;    -   hereditary motor and sensory neuropathy, autism, autosomal        dominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig's        disease, cancer, mitochondrial myopathy, diabetes mellitus and        deafness (DAD), Leber's hereditary optic neuropathy (LHON),        Leigh syndrome, subacute sclerosing encephalopathy, neuropathy,        ataxia, retinitis pigmentosa, and ptosis (NARP), myoneurogenic        gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with        ragged red fibers (MERRF), mitochondrial myopathy,        encephalomyopathy, lactic acidosis, stroke-like symptoms        (MELAS), mtDNA depletion, mitochondrial neurogastrointestinal        encephalomyopathy (MNGIE), dysautonomic mitochondrial myopathy,        mitochondrial channelopathy, or pyruvate dehydrogenase complex        deficiency (PDCD/PDH);    -   diabetic neuropathy;    -   chemotherapy-induced peripheral neuropathy; and    -   crush injury, spinal cord injury (SCI), traumatic brain injury,        stroke, optic nerve injury, and related conditions that involve        axonal disconnection.

Element 12: wherein the composition further comprises a pharmaceuticallyacceptable excipient.

Various changes could be made in the above methods without departingfrom the scope of the invention as defined in the claims below. It isintended that all matter contained in the above description, as shown inthe accompanying drawings, shall be interpreted as illustrative and notas a limitation.

What is claimed is:
 1. A method comprising: administering atherapeutically effective amount of a composition comprising one or moreof a mitofusin activator or a pharmaceutically acceptable salt thereofto a subject having or suspected of having a mitochondria-associateddisease, disorder or condition, the mitofusin activator having a formulaof

wherein: X is optionally substituted cycloalkyl or heterocycloalkyl; Zis optionally substituted pyridinyl or pyrimidinyl; R^(a) and R^(b) areindependently selected from the group consisting of H, F, alkyl, andC₃₋₇ cycloalkyl, or R^(a) and R^(b) taken together form a C₃₋₇cycloalkyl or heterocycloalkyl; R^(c) and R^(d) are independentlyselected from the group consisting of H, F, alkyl, and C₃₋₇ cycloalkyl,or R^(c) and R^(d) taken together form a C₃₋₇ cycloalkyl orheterocycloalkyl; Y is O, CR^(e)R^(f), or cycloalkyl; R^(e) and R^(f)are independently selected from the group consisting of H, F, alkyl, andcycloalkyl, or R^(e) and R^(f) taken together form C₃₋₇ cycloalkyl orheterocycloalkyl; o is 0 or 1; p is 1; and q is 0, 1, 2, 3, or 4,provided that if Y is cycloalkyl, the sum of o+p+q is not less than 3 orgreater than 5 and otherwise the sum of o+p+q is
 5. 2. The method ofclaim 1, wherein R^(a), R^(b), R^(c), R^(d), R^(e), and R^(f) are eachindependently H or alkyl.
 3. The method of claim 2, wherein Y iscyclopropyl or cyclobutyl.
 4. The method of claim 3, wherein o is
 0. 5.The method of claim 2, wherein X is


6. The method of claim 5, wherein Y is CH₂, and R^(a), R^(b), R^(c) andR^(d) are all H.
 7. The method of claim 2, wherein Z is 2-pyridinyl,3-pyridinyl, 4-pyridinyl, or 4-pyrimidinyl.
 8. The method of claim 7,wherein Y is CH₂, and R^(a), R^(b), R^(c) and R^(d) are all H.
 9. Themethod of claim 7, wherein the mitofusin activator is selected from thegroup consisting of


10. The method of claim 1, wherein the mitochondria-associated disease,disorder or condition is a peripheral nervous system (PNS) or centralnervous system (CNS) genetic or non-generic disorder, physical damage,and/or chemical injury.
 11. The method of claim 10, wherein the PNS orCNS disorder is selected from any one or a combination of: a chronicneurodegenerative condition wherein mitochondrial fusion, fitness, ortrafficking are impaired; a disease or disorder associated withmitofusin 1 (MFN1) or mitofusin 2 (MFN2) dysfunction; a diseaseassociated with mitochondrial fragmentation, dysfunction, ordysmotility; a degenerative neuromuscular condition such asCharcot-Marie-Tooth disease, Amyotrophic Lateral Sclerosis, Huntington'sdisease, Alzheimer's disease, Parkinson's disease; hereditary motor andsensory neuropathy, autism, autosomal dominant optic atrophy (ADOA),muscular dystrophy, Lou Gehrig's disease, cancer, mitochondrialmyopathy, diabetes mellitus and deafness (DAD), Leber's hereditary opticneuropathy (LHON), Leigh syndrome, subacute sclerosing encephalopathy,neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP),myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonicepilepsy with ragged red fibers (MERRF), mitochondrial myopathy,encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNAdepletion, mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), dysautonomic mitochondrial myopathy, mitochondrialchannelopathy, or pyruvate dehydrogenase complex deficiency (PDCD/PDH);diabetic neuropathy; chemotherapy-induced peripheral neuropathy; andcrush injury, spinal cord injury (SCI), traumatic brain injury, stroke,optic nerve injury, and related conditions that involve axonaldisconnection.
 12. A composition comprising one or more of a mitofusinactivator or a pharmaceutically acceptable salt thereof, the mitofusinactivator having a formula of

wherein: X is optionally substituted cycloalkyl or heterocycloalkyl; Zis optionally substituted pyridinyl or pyrimidinyl; R^(a) and R^(b) areindependently selected from the group consisting of H, F, alkyl, andC₃₋₇ cycloalkyl, or R^(a) and R^(b) taken together form a C₃₋₇cycloalkyl or heterocycloalkyl; R^(c) and R^(d) are independentlyselected from the group consisting of H, F, alkyl, and C₃₋₇ cycloalkyl,or R^(c) and R^(d) taken together form a C₃₋₇ cycloalkyl orheterocycloalkyl; Y is O, CR^(e)R^(f), or cycloalkyl; R^(e) and R^(f)are independently selected from the group consisting of H, F, alkyl, andcycloalkyl, or R^(e) and R^(f) taken together form C₃₋₇ cycloalkyl orheterocycloalkyl; o is 0 or 1; p is 1; and q is 0, 1, 2, 3, or 4,provided that if Y is cycloalkyl, the sum of o+p+q is not less than 3 orgreater than 5 and otherwise the sum of o+p+q is
 5. 13. The compositionof claim 12, wherein R^(a), R^(b), R^(c), R^(d), R^(e), and R^(f) areeach independently H or alkyl.
 14. The composition of claim 13, whereinY is cyclopropyl or cyclobutyl.
 15. The composition of claim 14, whereino is O.
 16. The composition of claim 13, wherein X is


17. The composition of claim 16, wherein Y is CH₂, and R^(a), R^(b),R^(c) and R^(d) are all H.
 18. The composition of claim 13, wherein Z is2-pyridinyl, 3-pyridinyl, 4-pyridinyl, or 4-pyrimidinyl.
 19. Thecomposition of claim 18, wherein Y is CH₂, and R^(a), R^(b), R^(c) andR^(d) are all H.
 20. The composition of claim 18, wherein the mitofusinactivator is selected from the group consisting of


21. The composition of claim 12, further comprising: a pharmaceuticallyacceptable excipient.